Du Pont graduated from Haverford School in 1957. He attended the University of Pennsylvania where he belonged to the Zeta Psi fraternity, and withdrew before completing his freshman year. Du Pont later attended college in Miami, Florida, where he studied under and was mentored by scientist Oscar T. Owre. He graduated from the University of Miami in 1965 with a Bachelor of Science degree in zoology. Du Pont went on to complete a doctorate in natural science from Villanova University in 1973.
During his graduate work, du Pont participated in several scientific expeditions to study and identify species of birds in the Philippines and South Pacific. As an ornithologist, du Pont is credited with the discovery of two dozen species of birds. He founded the Delaware Museum of Natural History in 1957. As a young man, he served on the board, helping guide the institution toward opening in 1972. After having been part of scientific expeditions, he served as director of the museum for many years.
Du Pont founded the Delaware Museum of Natural History in 1957, which opened to the public in 1972 on a site near Winterthur donated by his relative Henry Francis du Pont. John du Pont served on the board for many years. He also helped fund a new basketball arena at Villanova University, which opened in 1986. Originally it was called the John Eleuthère du Pont Pavilion, but after his conviction, his name was removed from the facility and simply called The Pavilion. Today the facility is named The Finneran Pavilion.