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).

He then toured with Violet Vanbrugh as Hugo in The Flame, and appeared at the London Playhouse in May 1924 as Langford in Leon Gordon’s White Cargo, in which he played all through 1924–25.

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.

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.

In 1934 he was reunited with Cornell on Broadway in Romeo and Juliet, playing Mercutio; Cornell was Juliet and Basil Rathbone was Romeo. In only ran 77 performances.

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 big hit. In 1935 Aherne and Cornell revived The Barretts of Wimpole Street on Broadway for 24 performances.

In 1937 he was Iago on Broadway to Walter Huston’s Othello.

Between 1939 and 1945, Aherne was married to actress Joan Fontaine; the marriage ended in divorce. He married Eleanor de Liagre Labrot in 1946, and their union lasted until his death (1986).

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 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.

In 1957 he went on a national tour of My Fair Lady playing Professor Henry Higgins. In 1960 he played the title role of The Trouble with Templeton of the American television anthology series The Twilight Zone.

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

In 1970, he appeared as a mystery guest on What’s My Line.