John Turturro was born in Brooklyn, New York, the son of Katherine 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.

Turturro was raised a Roman Catholic and moved to the Rosedale section of Queens, New York with his family, when he was six. He majored in Theatre Arts at the State University of New York at New Paltz, and completed his MFA at the Yale School of Drama.