Le Mesurier was born John Elton Le Mesurier Halliley, in Bedford on 5 April 1912. His parents were Charles Elton Halliley, a solicitor, and Amy Michelle (née Le Mesurier), whose family were from Alderney in the Channel Islands; both families were affluent, with histories of government Service or work in the legal profession. While John was an infant the family settled in Bury St Edmunds, in West Suffolk. He was sent to school, first to Grenham House in Kent, and later to Sherborne School in Dorset where one of his fellow-pupils was Alan Turing. Le Mesurier disliked both schools intensely, citing insensitive teaching methods and an inability to accept individualism. He later wrote: "I resented Sherborne for its closed mind, its collective capacity for rejecting anything that did not conform to the image of manhood as portrayed in the ripping yarns of a scouting manual".

From an early age Le Mesurier had been interested in acting and performing; as a child he had frequently been taken to the West End of London to watch Ralph Lynn and Tom Walls perform in the popular series of farces at the Aldwych Theatre. These experiences fuelled an early Desire to make a career on the stage. After leaving school he was initially persuaded to follow his father's line of work, as an articled clerk at Greene & Greene, a firm of solicitors in Bury St Edmunds; in his spare time he took part in local amateur dramatics. In 1933 he decided to leave the legal profession, and in September of that year enrolled at the Fay Compton Studio of Dramatic Art; a fellow-student was Alec Guinness, with whom he became close friends. In July 1934, the studio staged their annual public revue in which both Le Mesurier and Guinness took part; among the judges for the event were John Gielgud, Leslie Henson, Alfred Hitchcock and Ivor Novello. Le Mesurier received a Certificate of Fellowship, while Guinness won the Fay Compton prize. After the revue, rather than remain at the studio for further tuition Le Mesurier took an opportunity to join the Edinburgh-based Millicent Ward Repertory Players at a salary of £3.10s (£3.50) a week.

The Millicent Ward repertory company typically staged evening performances of three-act plays; the works changed each week, and rehearsals were held during the daytime for the following week's production. Under his birth name John Halliley, Le Mesurier made his stage debut in September 1934 at the Palladium Theatre, Edinburgh in the J. B. Priestley play Dangerous Corner, along with three other newcomers to the company. The reviewer for The Scotsman thought that Le Mesurier was well cast in the role. Appearances in While Parents Sleep and Cavalcade were followed by a break, as problems arose with the lease of the theatre. Le Mesurier then accepted an offer to appear with Alec Guinness in a John Gielgud production of Hamlet, which began in Streatham in the spring of 1935 and later toured the English provinces. Le Mesurier understudied Anthony Quayle's role of Guildenstern, and otherwise appeared in the play as an extra.

In July 1935, Le Mesurier was hired by the Oldham repertory company, based at the Coliseum Theatre; his first appearance with them was in a version of the Wilson Collison play, Up in Mabel's Room; he was sacked after one week for missing a performance after oversleeping. In September 1935, he moved to the Sheffield Repertory Theatre to appear in Mary, Mary, Quite Contrary, and also played Malvolio in Shakespeare's Twelfth Night. Le Mesurier later commented on the slow progress of his career: "had I known it was going to take so long, I might well have given the whole thing up". In 1937 he joined the Croydon Repertory Theatre, where he appeared in nine productions in 1936 and 1937. During this period Le Mesurier changed his professional name from John Halliley to John Le Mesurier; his biographer Graham McCann observes that "he never bothered, at least in public, to explain the reason for his decision". Le Mesurier used his new name for the first time in the September 1937 production of Love on the Dole.

Le Mesurier first appeared on television in 1938, thus becoming one of the medium's pioneering actors. His initial appearance was in a production of The Marvellous History of St Bernard in which he appeared as Seigneur de Miolans in a play adapted from a 15th-century manuscript by Henri Ghéon. Alongside the television appearance, he continued to appear on stage in Edinburgh and Glasgow with the Howard and Wyndham Players, at least until late 1938 when he returned to London and re-joined Croydon Repertory Theatre. His second spell with the troupe ended a few months later when, from May to October 1939 he appeared in Gas Light, first in London and subsequently on tour. The reviewer in The Manchester Guardian considered that Le Mesurier gave "a faultless performance", and that "the character is not overemphasised. One may praise it best by saying that Mr. Le Mesurier gives one a really uncomfortable feeling in the stomach".

In 1939, Le Mesurier accepted a role in the Robert Morley play Goodness, How Sad!, directed by June Melville—whose father Frederick owned a number of theatres, including the Lyceum, Prince's and Brixton. Melville and Le Mesurier soon began a romance, and were married in April 1940. Le Mesurier was conscripted into the army in September 1940; after his demobilisation in 1946, he discovered that his wife had become an alcoholic: "She became careless about appointments and haphazard professionally". As a result, the couple separated and were divorced in 1949.

In September 1940 Le Mesurier's rented home was hit by a German bomb, destroying all his possessions, including his call-up papers. In the same bombing raid, the theatre in Brixton in which he was working was also hit. A few days later he reported for basic training with the Royal Armoured Corps; in June 1941 he was commissioned into the Royal Tank Regiment. He served in Britain until 1943 when he was posted to British India where he spent the rest of the war. Le Mesurier later claimed that he had had "a comfortable war, with captaincy thrust upon me, before I was demobbed in 1946".

In June 1947, Le Mesurier went with fellow actor Geoffrey Hibbert to the Players' Theatre in London, where among the performers was Hattie Jacques. Le Mesurier and Jacques began to see each other regularly, although Le Mesurier was still married, albeit estranged from his wife. In 1949, when his divorce came through, Jacques proposed to Le Mesurier, asking him, "Don't you think it's about time we got married?". The couple married in November 1949 and had two sons, Robin and Kim.

On his return to Britain, Le Mesurier returned to acting, although he initially struggled for work, finding only a few minor roles. In February 1948 he made his film debut in the second feature comedy short Death in the Hand, which starred Esme Percy and Ernest Jay. He followed this with equally small roles in the 1949 film Old Mother Riley's New Venture—although his name was misspelt on the credits as "Le Meseurier"—and the 1950 crime film Dark Interval. During the same period he also frequently appeared on stage in Birmingham.

Le Mesurier undertook a number of roles on television in 1951, including that of Doctor Forrest in The Railway Children, the blackmailer Eduardo Lucas in Sherlock Holmes: The Second Stain, and Joseph in the nativity play A Time to be Born. In the same year Tony Hancock joined Le Mesurier's second wife, Hattie Jacques (the couple had married in 1949 following his divorce from June Melville earlier that year) in the radio series Educating Archie. Le Mesurier and Hancock became friends; they would often go for drinking sessions around Soho, where they ended up in jazz clubs. When Hancock left Educating Archie in 1954 to work on his own radio show, Hancock's Half Hour, he maintained his friendship with Le Mesurier, and Jacques joined the cast for the fourth series of Hancock's show, in 1956.

In 1952, as well as appearing in the films Blind Man's Bluff and Mother Riley Meets the Vampire, Le Mesurier also appeared as the Doctor in Angry Dust at the New Torch Theatre, London. Parnell Bradbury, writing in The Times, thought Le Mesurier had played the role extraordinarily well, although Harold Hobson, writing in The Sunday Times, thought that "the trouble with Mr. John Le Mesurier's Dr. Weston is that he approaches the man too snarlingly ... [it is] a notion of genius that would be unacceptable anywhere outside Victorian melodrama". In 1953, he had a role as a bureaucrat in the short film The Pleasure Garden, which won the Prix de Fantasie Poetique at the Cannes Film Festival in 1954. After a long run of small roles in second features, his 1955 portrayal of the registrar in Roy Boulting's comedy Josephine and Men, "jerked him out of the rut", according to Philip Oakes.

Following his appearance in Josephine and Men, John and Roy Boulting cast Le Mesurier as a Psychiatrist in their 1956 Second World War film, Private's Progress. The cast featured many leading British actors of the time, including Ian Carmichael and Richard Attenborough. Dilys Powell, reviewing for The Sunday Times, thought that the cast was "embellished" by Le Mesurier's presence, among others. Later in 1956 Le Mesurier again appeared alongside Attenborough, with small roles in Jay Lewis's The Baby and the Battleship and Roy Boulting's Brothers in Law, the latter which also featured Carmichael and Terry-Thomas. He was also active in television, in a variety of roles in episodes of Douglas Fairbanks Presents, a series of short dramas.

Le Mesurier's friendship with Tony Hancock provided a further source of work when Hancock asked him to be one of the regular supporting actors in Hancock's Half Hour, when it moved from radio to television. Le Mesurier subsequently appeared in seven episodes of the show between 1957 and 1960, and then in two episodes of a follow-up series entitled Hancock. In 1958 he appeared in ten films, among them Roy Boulting's comedy Happy Is the Bride, about which Dilys Powell wrote in The Sunday Times: "[M]y vote for the most entertaining contributions ... goes to the two fathers, John Le Mesurier and Cecil Parker". In 1959, the busiest year of his career, Le Mesurier took part in 13 films, including I'm All Right Jack, which was critically and commercially the most successful of Le Mesurier's credited films that year, although he also had an uncredited role as a Doctor in Ben-Hur.

Le Mesurier appeared in nine films in 1960, as well as nine television programmes, including episodes of Hancock's Half Hour, Saber of London and Danger Man. His work the following year included a part in Peter Sellers's directorial debut Mr. Topaze, a film which failed both critically and commercially. He provided the voice of Mr. Justice Byrne in a recording of excerpts from the transcript of R v Penguin Books Ltd.—the court case concerning the publication of D. H. Lawrence's Lady Chatterley's Lover—which also featured Michael Hordern and Maurice Denham. J.W. Lambert, reviewing for The Sunday Times, wrote that Le Mesurier gave "precisely the air of confident incredulity which the learned gentleman exhibited in court". Later that year he played Hancock's office manager in the first of Tony Hancock's two principal film vehicles, The Rebel.

Jacques began an affair in 1962 with her driver, John Schofield, who gave her the attention and support that Le Mesurier did not. When Jacques decided to move Schofield into the family home, Le Mesurier moved into a separate room and tried to repair the marriage. He later commented about this period: "I could have walked out, but, whatever my feelings, I loved Hattie and the children and I was certain—I had to be certain—that we could repair the damage". The affair caused a downturn in his health; he collapsed on holiday in Tangier in 1963 and was hospitalised in Gibraltar. He returned to London to find the situation between his wife and her lover was unchanged, which caused a relapse.

In a change from his usual comedic roles, Le Mesurier portrayed the Reverend Jonathan Ives in Jacques Tourneur's 1965 science fiction film, City Under the Sea, before returning to comedy in Where the Spies Are, a comedy-adventure film directed by Val Guest, which starred David Niven. In 1966 Le Mesurier also played the role of Colonel Maynard in the ITV sitcom George and the Dragon, with Sid James and Peggy Mount. The programme ran to four series between 1966 and 1968, totalling 26 episodes. He also took a role in four episodes of a Coronation Street spin-off series, Pardon the Expression, in which he starred opposite Arthur Lowe.

During the final stages of the breakdown of his marriage, Le Mesurier met Joan Malin at the Establishment club in Soho in 1963. The following year he moved out of his marital house, and that day proposed to Joan, who accepted his offer. Le Mesurier allowed Jacques to bring a divorce suit on grounds of his own infidelity, to ensure that the press blamed him for the break-up, thus avoiding any negative publicity for Jacques. Le Mesurier and Malin married in March 1966. A few months after they were married, Joan began a relationship with Tony Hancock, and left Le Mesurier to move in with the Comedian. Hancock was a self-confessed alcoholic by this time, and was verbally and physically abusive to Joan during their relationship. After a year together, with Hancock's violence towards her worsening, Joan attempted suicide; she subsequently realised that she could no longer live with Hancock and returned to her husband. Despite this, Le Mesurier remained friends with Hancock, calling him "a comic of true genius, capable of great warmth and generosity, but a tormented and unhappy man". Without Le Mesurier's knowledge, Joan resumed her affair with Hancock and, when the comic moved to Australia in 1968, she planned to follow him if he was able to overcome his alcoholism. She abandoned these plans and remained with Mesurier after Hancock committed suicide on 25 June 1968.

In between the annual shooting of Dad's Army, Le Mesurier acted in films, including the role of the prison governor opposite Noël Coward in the 1969 Peter Collinson-directed The Italian Job. The cinema Historian Amy Sargeant likened Le Mesurier's role to the "mild demeanour" of his Sergeant Wilson character. In 1970, Le Mesurier appeared in Ralph Thomas's Doctor in Trouble as the purser; he also made an appearance in Vincente Minnelli's On a Clear Day You Can See Forever, a romantic fantasy musical.

In 1971 Le Mesurier played the lead role in Dennis Potter's television play Traitor, in which he portrayed a "boozy British aristocrat who became a spy for the Soviets"; his performance won him a British Academy of Film and Television Arts "Best Television Actor" award. Writing for the British Film Institute, Sergio Angelini considered "Le Mesurier is utterly compelling throughout in an atypical role". Chris Dunkley, writing in The Times, described the performance as "a superbly persuasive portrait, made vividly real by one of the best performances Mr Mesurier [sic] has ever given". The reviewer for The Sunday Times agreed, saying that Le Mesurier, "after a lifetime supporting other actors with the strength of a pit-prop, gets the main part; he looks, sounds and feels exactly right". Reviewing for The Guardian, Nancy Banks-Smith called the role "his Hamlet", and said that it was worth waiting for. Although delighted to have won the award, Le Mesurier commented that the aftermath proved "something of an anticlimax. No exciting offers of work came in".

Le Mesurier made a cameo appearance in Val Guest's 1972 sex comedy Au Pair Girls, and starred alongside Warren Mitchell and Dandy Nichols in Bob Kellett's The Alf Garnett Saga. In 1974 he played a police inspector in a similar Val Guest comedy, Confessions of a Window Cleaner, alongside Robin Askwith and Antony Booth. The following year he also narrated Bod, an animated children's programme from the BBC; there were thirteen episodes in total.

Le Mesurier was a heavy drinker, but was never noticeably drunk. In 1977 he collapsed in Australia and flew home, where he was diagnosed with cirrhosis of the liver and ordered to stop drinking. Until then he had not considered himself an alcoholic, although he accepted that "it was the cumulative effect over the years that had done the damage". It was a year and a half before he drank alcohol again, when he avoided spirits and drank only beer. Jacques claimed that his calculated vagueness was the result of his dependence on cannabis, although according to Le Mesurier the drug was not to his taste; he smoked it only during his period of abstinence from alcohol. Le Mesurier's favoured pastime was visiting the jazz clubs around Soho, such as The Establishment or Ronnie Scott's, and he observed that "listening to artists like Bill Evans, Oscar Peterson or Alan Clare always made life seem that little bit brighter".

Le Mesurier played The Wise Old Bird in the 1980 BBC Radio 4 series The Hitchhiker's Guide to the Galaxy and appeared on the same station as Bilbo Baggins in the 1981 radio version of The Lord of the Rings. In the spring of 1980 he took the role of David Bliss alongside Constance Cummings—as Judith Bliss—in a production of Noël Coward's 1920s play Hay Fever. Writing for The Observer, Robert Cushman thought that Le Mesurier played the role with "deeply grizzled torpor", while Michael Billington, reviewing for The Guardian, saw him as a "grey, gentle wisp of a man, full of half-completed gestures and seraphic smiles".

He took on the role of Father Mowbray in Granada Television's 1981 adaptation of Brideshead Revisited. He guest-starred in episodes of the British comedy television series The Goodies, and in an early episode of Hi-de-Hi!. His final film appearance was also Peter Sellers's final cinema role, The Fiendish Plot of Dr. Fu Manchu, which was completed just months before Sellers's death in July 1980.

In 1982 Le Mesurier reprised the role of Arthur Wilson for It Sticks Out Half a Mile, a radio sequel to Dad's Army, in which Wilson had become bank manager of the Frambourne-on-Sea branch, while Arthur Lowe's character, Captain George Mainwaring, was trying to apply for a loan to renovate the local pier. The death of Lowe in April 1982 meant that only a pilot episode was recorded, and the project was suspended. It was revived in 1982 with Lowe's role replaced by two other Dad's Army cast members: Pike, played by Ian Lavender, and Hodges, played by Bill Pertwee. A pilot and twelve episodes were subsequently recorded, and broadcast in 1984. Le Mesurier also teamed up with another ex-Dad's Army colleague, Clive Dunn, to record a novelty single, "There Ain't Much Change from a Pound These Days"/"After All These Years", which had been written by Le Mesurier's stepson, David Malin. The single was released on KA Records in 1982.

Towards the end of his life Le Mesurier wrote his autobiography, A Jobbing Actor; the book was published in 1984, after his death. Le Mesurier's health visibly declined from July 1983 when he was hospitalised for a short time after suffering a haemorrhage. When the condition recurred later in the year he was taken to Ramsgate Hospital; after saying to his wife, "It's all been rather lovely", he slipped into a coma and died on 15 November 1983, aged 71. His remains were cremated, and the ashes buried at the Church of St. George the Martyr, Church Hill, Ramsgate. His epitaph reads: "John Le Mesurier. Much loved actor. Resting." His self-penned death notice in The Times of 16 November 1983 stated that he had "conked out" and that he "sadly misses family and friends".

The Guardian reflected on Le Mesurier's popularity, observing that "No wonder so many whose lives were very different from his own came to be so enormously fond of him". A memorial Service was held on 16 February 1984 at the "Actors' Church", St Paul's, Covent Garden, at which Bill Pertwee gave the eulogy.

Le Mesurier's second and third marriages have been the subject of two BBC Four biographical films, the 2008 Hancock and Joan on Joan Le Mesurier's affair with Tony Hancock—with Le Mesurier played by Alex Jennings—and the 2011 Hattie on Jacques's affair with John Schofield—with Le Mesurier played by Robert Bathurst. In We're Doomed! The Dad's Army Story, a 2015 comedy drama about the making of Dad's Army, Le Mesurier was portrayed by Julian Sands.