Turpin was born in New Orleans, Louisiana on September 19, 1869, the son of a candy store owner, Ernest Turpin, and Sarah Buckley.

Ben Turpin first appeared on film in 1907 for Essanay Studios in Chicago in various small parts and comic bits. In addition to his on-screen work, he worked as a carpenter and janitor for Essanay. In the 1909 film Mr. Flip, Turpin receives what is believed to have been the first pie-in-the-face. By 1912 he was an established – if not major – screen personality, giving interviews and writing articles for the new fan magazines (the first of which had started the year before).

Charlie Chaplin joined the Essanay company in 1915, and the studio made Turpin his second Banana. Chaplin was maturing as a filmmaker, working slowly and intuitively. Turpin, however, was impatient with Chaplin's methods. The earthy Turpin understood straightforward slapstick more than comic subtlety. The Chaplin-Turpin duo didn't last long, with Chaplin abandoning Chicago for California. Turpin does share one additional credit with Chaplin: after Chaplin filmed Burlesque on Carmen in two reels, Essanay filmed new scenes with Turpin to pad the picture into a featurette, doubling its length.

In 1917 Ben Turpin joined the leading comedy company, the Mack Sennett studio. Turpin's aptitude for crude slapstick suited the Sennett style perfectly, and Sennett's Writers often cast the ridiculous-looking Turpin against type (a rugged Yukon miner; a suave, worldly lover; a stalwart cowboy; a fearless stuntman, etc.) for maximum comic effect. Through the 1920s his roles often spoofed serious actors and celebrities of the time – e.g., "The Shriek" for "The Sheik" – and Turpin became one of film's most popular comics. Turpin appeared in both short subjects and feature films for Sennett. Delighted with his success, he took to introducing himself with the phrase, "I'm Ben Turpin; I make $3,000 a week."

Turpin famously bought a $25,000 insurance policy with Lloyd's of London, payable if his eyes ever uncrossed. A 1920 version of the story had his eyes insured for $100,000. How serious this was is open to question; such publicity Stunts centered around a performer's "trademark" were Common at the time.

Sennett terminated most of his staff's contracts in 1928, and closed the studio to retool for the new talking pictures. Turpin was signed by the low-budget Weiss Brothers-Artclass company, perhaps the most ambitious coup that Artclass ever attempted. Turpin made two-reel comedies there for one year. Artclass usually traded on his peculiar vision with titles like Idle Eyes and The Eyes Have It.

1929 saw many silent-film stars uncertain about their Future employment, with the new talking pictures requiring new skills and techniques. Ben Turpin chose to retire. He had invested his earnings in real estate, and, being highly successful at this, had no financial need for more work. Producers soon sought him out for gag appearances in films. He commanded a flat fee of $1000 per appearance, regardless of whether it was a speaking role or a fleeting cameo. Among the most memorable of these cameos was in Paramount's Million Dollar Legs (1932) starring W. C. Fields, Jack Oakie, and Susan Fleming.

He starred in only one more short subject, Keystone Hotel (Warner Bros., 1935), a two-reel reunion of silent-era comedians. Turpin's speaking voice was a gritty rasp that retained elements of the New Orleans "Yat" accent of his youth. His last feature film was Laurel and Hardy's Saps at Sea in 1940, in which his cross-eyed face served as a joke punchline. He was paid his $1000 for one quick shot of his face and just 16 words of dialogue. Death prevented his scheduled appearance in Charlie Chaplin's The Great Dictator.

Turpin and Sennett both appeared as themselves (in Technicolor) in Hollywood Cavalcade (1939), a partly fictionalized movie about the silent-film era. This movie contains a sequence in which Turpin reports for work and prepares to go onto the set in character. In the dressing room he picks up a hand mirror and checks his reflection as he deliberately crosses his eyes as extremely as possible. In this sequence, it can be seen that Turpin's left eye was actually normal when he was not performing, and that he intentionally crossed it (to match his misaligned right eye) as part of his screen character.

Ben Turpin died July 1, 1940 of a heart attack and was interred in the Forest Lawn Memorial Park Cemetery in Glendale, California, following a Requiem Mass at the Church of the Good Shepherd in Beverly Hills. He was eulogized as "a fine member of his church, strong in his faith" by Father J. P. Concannon. His pallbearers included Andy Clyde, Billy Bevan, James Finlayson, and Charlie Murray.

In the film The Comic (1969), Mickey Rooney plays a fictional silent-film Comedian named "Cockeye Van Buren", who is genuinely cross-eyed. Although this character does not otherwise resemble Turpin, the handicap given to Rooney's role is clearly inspired by Turpin.