Macnee, the older of two sons (he has a younger brother, James), was born Daniel Patrick Macnee in Paddington, London, the son of Dorothea Mary (née Henry) and Daniel "Shrimp" Macnee, a race horse trainer. His maternal grandmother was Frances Alice Hastings, who descended from the Earls of Huntingdon - Macnee has long suggested that he may be a distant relation of Robin Hood, sometimes said to have been a black sheep of the Huntingdon family. Macnee's great-grandfather was Scottish portrait artist Sir Daniel Macnee and he is a cousin of the magician David Nixon.
His parents divorced after his mother declared her lesbianism and had a live-in partner (referred to in Macnee's memoirs as "Uncle Evelyn") who helped pay for young Patrick's schooling. He was educated at Eton College, was commissioned as a Lieutenant in the Royal Navy and was awarded the Atlantic Star for his service during World War II. After nurturing his acting career in Canada, Macnee appeared in supporting roles in a number of films, notably in the Gene Kelly vehicle Les Girls (as an Old Bailey barrister) and opposite Anthony Quayle in the 1956 war movie The Battle of the River Plate. He had a small role in the 1951 version of Scrooge (A Christmas Carol in the U. S.) as young Jacob Marley.
Despite numerous roles in theatre, on television and in cinema, Macnee is still best known as John Steed in the series The Avengers (broadcast from 1961 to 1969). Initially a secondary character — the series was conceived as a vehicle for Ian Hendry, who played an associate of Steed's — Steed (and Macnee) became the centre of the show after Hendry's departure at the end of the first season. He played opposite a succession of female partners that included Honor Blackman, Diana Rigg, and finally Linda Thorson. Steed was also the central character of a revival, The New Avengers, in which he was teamed with Purdey, portrayed by Joanna Lumley, and Mike Gambit, played by Gareth Hunt.
Although Macnee evolved the role as the series progressed, the key elements of Steed's persona and appearance were there from very early on: the slightly mysterious demeanour, and increasingly, the light, suave, flirting tone with ladies (and always with his female assistants). Finally, from the episodes with Honor Blackman onwards, the trademark bowler hat and umbrella completed the image. Traditionally associated with London 'city gents', the suit, umbrella and bowler had developed in the post-war years as mufti for ex-servicemen attending Armistice Day ceremonies. Macnee, alongside designer Pierre Cardin, adapted the look into a style all his own, and he went on to design several outfits himself for Steed based on the same basic theme.
During the 1960s, Macnee co-wrote two original novels based upon The Avengers: Dead Duck and Deadline. In 1988, he wrote his autobiography entitled Blind in One Ear. In 1995, he hosted a documentary, The Avengers: The Journey Back, directed by Clyde Lucas.
When asked in June 1982 which Avengers female lead was his favourite, Macnee declined to give a specific answer. "Well, I'd rather not say. To do so would invite trouble," he told TV Week magazine. Macnee did provide his evaluation of the female leads. Of Honor Blackman he said "She was wonderful, presenting the concept of a strong-willed, independent and liberated woman just as that sort of woman was beginning to emerge in society." Diana Rigg was "One of the world's great actresses. A superb comedienne. I'm convinced that one day she'll be Dame Diana." Linda Thorson was "one of the sexiest women alive" while Joanna Lumley was "superb in the role of Purdey. An actress who is only now realising her immense potential." 
Macnee's other notable roles have included playing 'Sir Godfrey Tibbett' opposite Roger Moore in the James Bond movie A View to a Kill, as 'Major Crossley' in The Sea Wolves (again with Moore), guest roles in Alias Smith and Jones, Hart to Hart, Murder, She Wrote, Battlestar Galactica and The Love Boat. Though Macnee found fame as the heroic Steed, the majority of his guest appearances have been in villainous roles. He also presented the American paranormal series, Mysteries, Magic and Miracles. Macnee made his Broadway debut as the star of Anthony Shaffer's mystery Sleuth in 1972 and subsequently headlined the national tour of that play.  .
Macnee had recurring roles in the crime series Gavilan with Robert Urich and in the 1984 satire on big business, Empire as the menacing M.D. 'Calvin Cromwell'. In 1984, Macnee appeared in Magnum, P.I. as a retired British agent who believes he is Sherlock Holmes (in a season 4 episode entitled Holmes Is Where The Heart Is). He in fact had played Dr Watson to Roger Moore's Sherlock Holmes in a 1976 TV movie, Sherlock Holmes in New York and went on to play Holmes in another TV movie, The Hound of London (1993). He played Watson in two TV movies with Christopher Lee (Incident at Victoria Falls and Sherlock Holmes and the Leading Lady).
He also appeared in several cult movies: in The Howling as 'Dr George Waggner' (named whimsically after the director of 1941's The Wolf Man) and as 'Sir Denis Eton-Hogg' in the rockumentary comedy This is Spinal Tap. He took over Leo G. Carroll's role as the head of U.N.C.L.E. in The Return of The Man from U.N.C.L.E. in 1983. Patrick starred in the 1990s science fiction series Super Force as E.B. Hungerford (called "MR. H." by lab assistant F.X.) in the pilot and his computer counter-part; after his character was killed. Macnee also appeared as a supporting character in the 1989 science fiction parody, Lobster Man from Mars, as Prof. Plocostomos.
Macnee serves as the narrator for several "behind-the-scenes" featurettes featured on the James Bond series of DVDs. He lent his voice in a cameo as 'Invisible Jones' in the 1998 critically lambasted film version of The Avengers (in which Steed was played by Ralph Fiennes), and he also featured in two pop videos: in his Steed persona in The Pretenders' video Don't Get Me Wrong, and in the Oasis' video of their song Don't Look Back in Anger in 1996, with the familiar smart suit and umbrella, but minus the bowler hat. Macnee was the voice of the evil Imperious Leader of the Cylons in the original 1970's version of Battlestar Galactica, and appeared onscreen as Count Iblis - another manifestation of the Imperious Leader.
Macnee has two children, Rupert and Jenny, from his first marriage to Barbara Douglas (from 1942 to 1956). His second marriage (1965 – 1969) was to actress Katherine Woodville. He is married to third wife Baba Majos de Nagyzsenye (1988-).
Macnee became an American citizen in 1959.