York was born in Muskogee, Oklahoma. The son of a dentist who died when York was 15, York grew up in Little Rock and attended local schools until college. He graduated from the University of Notre Dame in South Bend in 1971. He received a doctorate in blood pathology from Stritch School of Medicine at Loyola University Chicago in 1975, and engaged in seven years of post-doctoral study, specializing in the laboratory diagnosis of lymphoma and leukemia.
York left academia to establish DeYor Laboratories in Boardman, Ohio, a partnership with his father-in-law, Edward J. DeBartolo Sr. In 1999, he sold DeYor Laboratories. York has made a number of contributions in the medical field, having given a $1.5 million contribution to establish an Endowed Chair in Pathology and an Endowed Chief Residency in Pathology at the University of Arkansas for Medical Sciences. In 1999, he donated a science and math building to Catholic High School for Boys, his alma mater.
On December 28, 2008 John and Denise York announced that their son, Jed York, would become the Team President and Owner and that they would become co-chairmen responsible for providing resources and maintaining their role of interacting with other owners and NFL executives. John continues to serve as Chairman of the NFL's International Committee and also sits on the league's Audit and Business Ventures Committees.