John Turturro was born in Brooklyn, New York, the son of Katherine Florence (Incerella) and Nicholas Turturro. His mother was born in the U.S., to Italian parents with roots in Sicily, and was an amateur jazz singer, who had worked in a naval yard during World War II. His father had immigrated to the United States from Giovinazzo, Italy at age six and later worked as a carpenter and construction worker before joining the U.S. Navy during the war. Serving as a Sailor, he was with the D-Day fleet supporting the landing operations of Allied troops in Normandy, France, in 1944.

Turturro's first film appearance was a non-speaking extra role in Martin Scorsese's critically acclaimed Raging Bull (1980). He created the title role of John Patrick Shanley's Danny and the Deep Blue Sea at the Playwrights Conference at the Eugene O'Neill Theatre Center in 1983. He repeated it the following year Off-Broadway and won an Obie Award.

Turturro's brother is actor Nicholas Turturro. Artist Ralph Turturro, Composer and film Director Richard Termini, and Actress Aida Turturro are his cousins. He has two sons: Amedeo (born 1990) and Diego (born 2000), with his wife, Actress Katherine Borowitz.

Turturro hosted Saturday Night Live in 1994, where he spoofed his then-recently made film, Quiz Show, being told he was ineligible to host unless he answered questions in a booth and if he failed, the honor of hosting would go to Joey Buttafuoco, who was actually backstage to witness Turturro's test. He won an Emmy award for his portrayal of Adrian Monk's brother Ambrose in the USA Network series Monk, and reprised the role on numerous occasions. He has also been nominated and won many awards from film organizations such as Screen Actors Guild, Cannes Film Festival, Golden Globes and others.

Turturro produced and directed, as well as acted in, the film Illuminata (1999), which also starred his wife, Actress Katherine Borowitz. He wrote and directed the film Romance and Cigarettes (2005). In 2006 he appeared in Robert De Niro's The Good Shepherd, and as the Sector 7 agent Simmons in four films of the Transformers live-action series. In 2010, he directed (and had cameo on-screen appearances in) Passione, which chronicles the rich musical heritage of Naples, Italy.

John Turturro participates as a member of the Jury for the New York International Children's Film Festival (NYICFF), which is dedicated to screening films for children between the ages of 3 and 18. In January 2011, Turturro received his Italian passport, and holds dual Italian and U.S. citizenship.

Turturro's fifth directorial film Fading Gigolo premiered at the Toronto International Film Festival (TIFF) in mid-September 2013. Turturro also acts in the film alongside Woody Allen, who played a novice pimp overseeing the sex work of Turturro's character. During a September 2013 interview, Turturro expressed his intention to draw parallels between sex work and acting, explaining that the latter is a "service business" in which actors are "acting out people's wishes or fantasies." In March 2014, Turturro received the Career Achievement tribute and award at the 31st Edition of the Miami International Film Festival at the Olympia Theater in Downtown Miami.

Turturro has appeared in both comedy and drama films, and engaged in an extended collaboration with the Coen Brothers—he appeared in their films Miller's Crossing (1990), Barton Fink (1991), The Big Lebowski (1998), and O Brother, Where Art Thou? (2000). He has also appeared in several of Adam Sandler's movies, such as Mr. Deeds (2002) and You Don't Mess with the Zohan (2008). He played a severely disturbed patient of Jack Nicholson's character in the comedy Anger Management and played Johnny Depp's character's antagonist in Secret Window.