Aherne died of heart failure in Venice, Florida, USA at the age of 83. He was honoured with a star on the Hollywood Walk of Fame at 1772 Vine Street.

He first appeared on the stage in Birmingham with the Pilgrim Players (which subsequently developed into the Birmingham Repertory Theatre), on 5 April 1910, in Fifinella; and made his first appearance on the London stage at the Garrick Theatre, 26 December 1913, in Where the Rainbow Ends, a fairy play by Clifford Mills and John Ramsey, with music by Roger Quilter, which ran at various theatres for over 25 years.

He then studied with a view to becoming an Architect, but, having had considerable amateur experience in Birmingham, and with Liverpool's Green Room Club, he obtained an engagement under Robert Courtneidge, and appeared at London's Savoy Theatre, opening on 26 December 1923, as Jack O'Hara in a revival of Paddy the Next Best Thing, the play by W. Gayer-Mackay and Robert Ord (from the novel).

Aherne's first screen appearance was in the crime film The Eleventh Commandment in 1924. He made several appearances in productions at Cricklewood Studios by Stoll Pictures, then the largest British film company, including two directed by Sinclair Hill, The Squire of Long Hadley (1925) and A Woman Redeemed (1927). He was also in King of the Castle (1925), and the comedy Safety First (1926).

In 1926 he accompanied Dion Boucicault, Jr. to Australia, where he appeared in several plays by J.M. Barrie: as Valentine Brown in the comedy Quality Street, John Shand in the comedy What Every Woman Knows, Crichton in The Admirable Crichton, Simon and Harry in Mary Rose; and Willocks in Aren't We All? another comedy by Frederick Lonsdale.

Aherne reappeared in London at the Strand in March 1927, again as Langford in White Cargo, and continued on the London stage in a succession of plays until late 1930 when he went to America.

His final silents were two films Shooting Stars and Underground by the rising Director Anthony Asquith. Aherne made his talkie debut in The W Plan (1930) directed by Victor Saville. He appeared opposite Madeleine Carroll in Madame Guillotine (1931).

Aherne made his first appearance on the New York City stage at the Empire Theatre on 9 February 1931, playing Robert Browning in Rudolph Besier's play The Barretts of Wimpole Street opposite Katharine Cornell. The play was a big success, running for 370 performances. Miss Cornell and Aherne remained lifelong friends and he played in many of her subsequent productions.

Aherne returned to Broadway in 1932 for Lucrece which starred Cornell. It only had a short run. He then went to Hollywood where he made his American film debut in The Song of Songs (1933) with Marlene Dietrich.

He returned to England where he starred in the film of Basil Dean's The Constant Nymph (1933).

Aherne went to MGM where he co-starred with Helen Hayes in What Every Woman Knows (1934). He stayed at that studio to support Joan Crawford in I Live My Life (1935), which was a bit hit. In 1935 Aherne and Cornell revived The Barretts of Wimpole Street on Broadway for 24 performances.

At RKO Aherne was in Sylvia Scarlett (1935) with Katharine Hepburn and Cary Grant, a notorious flop. Aherne went back to Broadway for Cornell's production of Saint Joan (1936), co-starring Maurice Evans. He returned to Hollywood for Beloved Enemy (1936) with Merle Oberon at Goldwyn Productions.

Aherne was top billed in The Great Garrick (1937), directed by James Whale at Warners. He supported Constance Bennett in Merrily We Live (1938) for Hal Roach Studios. He was Oscar-nominated for his role as Emperor Maxmilian in Juarez (1939).

Between 1939 and 1945, Aherne was married to Actress Joan Fontaine, which ended in divorce. He then married Eleanor de Liagre Labrot. He was the younger brother of actor Pat Aherne.

Aherne was billed over Rita Hayworth in The Lady in Question (1940) at Columbia. He made Hired Wife (1940) at Universal with Rosalind Russell; for that studio he did The Man Who Lost Himself (1941) with Kay Francis.

MGM put Aherne in support of Jeanette MacDonald for Smilin' Through (1941). He supported Claudette Colbert in Skylark (1941) at Paramount and Rosalind Russell in My Sister Eileen (1942) at Columbia. He stayed at that studio to support Loretta Young in A Night to Remember (1942) and was one of many stars in Forever and a Day (1943).

In 1943 he quit films to become a FLIGHT instructor for the air force in Arizona. In November 1943 it was reported Columbia paid him $144,958 for the year, making him the second highest paid person at Columbia, after Harry Cohn.

He fell ill with influenza while touring army camps in 1944.

In 1945 he and Cornell returned to Broadway in a revival of The Barretts of Wimpole Street. He stayed in New York to appear in The French Touch (1945–46) directed by René Clair.

Aherne returned to movies with RKO's The Locket (1946), billed after Laraine Day. He was top billed in Smart Woman (1948), co-starring Producer Constance Bennett. He did Drums Along the Amazon (1948) for Republic.

Aherne was in a Broadway revival of She Stoops to Conquer (1949–50).

Aherne made his television debut with "Dear Brutus" for The Ford Theatre Hour (1950), which he had performed on stage in Boston. He followed it with "The Magnificent Gesture" for Armstrong Circle Theatre (1950), "A Well-Remembered Voice" for Lux Video Theatre, "The Old Flame" for The Billy Rose Show (1951), "The Buccaneer" for Pulitzer Prize Playhouse (1951), and Betty Crocker Star Matinee (1952).

He and Cornell were reunited on stage in The Constant Wife (1951–52) then Aherne returned to Hollywood. He had support roles in I Confess (1953) directed by Alfred Hitchcock and Titanic (1953) (as Captain E.J. Smith).

Aherne co-starred in the Florence Nightingale episode of Theatre Guild on the Air 13 April 1952. In 1945, he played sleuth Simon Templar in the mystery series, The Saint.

Aherne did Escapade (1953) on Broadway and "Two for Tea" for Lux Video Theatre and "Element of Risk" and "Breakdown" for Robert Montgomery Presents (1953).

He did Quadrille (1954–55) on Broadway with the Lunts then "Now in Rehearsal" for The Eddie Cantor Comedy Theater (1955). Aherne did "The Martyr" for General Electric Theater (1955), "Reunion in Vienna" for Producers' Showcase (1955), and "The Round Dozen" and "Appearances and Reality" for The Star and the Story (1955).

Aherne went to MGM for The Swan (1956). On TV he did "One Minute from Broadway" for Sneak Preview (1956), "Night Shriek" for Climax! (1956), "The Sacred Trust" and "The Lamp of Father Cataldo" for Crossroads (1956), "The Transfer" for The Errol Flynn Theatre (1956), "Safe Enough" for Studio 57 (1957), "Story Without a Moral" for Goodyear Theatre (1959).

In 1957 he went on a national tour of My Fair Lady playing Professor Henry Higgins.

Aherne's final Broadway appearance was in Dear Liar (1960) with Cornell, where he played George Bernard Shaw ("with great vivacity" according to the New York Times) opposite Cornell's Mrs Patrick Campbell. He was in "The Trouble with Templeton" for The Twilight Zone (1960) and the film Susan Slade (1961). He did "The Bruce Saybrook Story" on Wagon Train (1961), and "The Gentleman's Gentleman" on Rawhide (1961). He also appeared as guest host on the TV panel show The Name's the Same.

Aherne's final film roles included Lancelot and Guinevere (1963) as King Arthur, The Waltz King (1964) for Disney (as Johann Strauss I), and The Cavern (1964).

He settled in Switzerland. He appeared in a play in England and agreed to return to Hollywood to play Rosalind Russell's love interest in Rosie! (1967).

Aherne published his autobiography A Proper Job in 1969, as well as A Dreadful Man (1979), a biography of his friend George Sanders.