Nitschke was born in Elmwood Park, Illinois. He was orphaned at age thirteen, when his father died (his mother died when Ray was three), and was raised by an older brother. He played quarterback at Proviso East High School in Maywood, Illinois, guiding his team to a suburban league title, and made the All-State team. In 1954, Nitschke accepted a football scholarship to the University of Illinois, where he switched to fullback in his sophomore year, scoring four touchdowns on long runs against Iowa State. While a bruising runner, his greatest strength was as a linebacker on the defensive side of the ball.

He was selected, at age 20, in the third round of the 1958 NFL draft, the 36th overall pick. This draft, held on December 2, 1957, included two other significant Packers of the 1960s: fullback Jim Taylor of LSU (2nd rd., 15th overall) and right guard Jerry Kramer of Idaho (4th rd., 39th overall). Their rookie season in 1958 was dismal, recording just one win (and one tie); finishing with the worst record in the 12 team league.

A month after the 1958 season ended, Vince Lombardi was hired as head coach. Nitschke became a full time starter in 1962, the anchor of a disciplined defense that helped win five NFL titles and the first two Super Bowls in the 1960s. He was the MVP of the 1962 NFL Championship game, accepting the prize of a 1963 Chevrolet Corvette. He was an All-Pro three consecutive seasons (1964-66), and was elected to the Pro Football Hall of Fame in 1978. He died twenty years later, at age 61, of an apparent heart attack while visiting family and friends in Venice, Florida.

Nitschke appeared in the 1968 film Head and the 1974 film The Longest Yard.

His #66 is only of only five numbers retired by the Packers, and in his honor the team has named one of its two outdoor practice fields Ray Nitschke Field.