Van Hooff studied biology at Utrecht University. After reading the book The Expression of the Emotions in Man and Animals of Charles Darwin he decided he wished to do research on the facial expressions of primates. Not having an opportunity to do so at Utrecht University, he was helped by professor of comparative physiology Sven Dijkgraaf to study at the University of Oxford under Nikolaas Tinbergen and Desmond Morris. In 1971 he obtained his doctorate with a dissertation titled:"Aspecten van het sociale gedrag en de communicatie bij humane en hogere niet-humane primaten" under Nico Frijda and Dijkgraaf. During the 1960s the parents of Van Hooff needed more help with running the zoo, as Jan was studying in the United Kingdom his brother Antoon van Hooff helped and later became director.
In 1966 Van Hooff was involved in research concerning socialization of a large group of chimpanzees held at a United States Air Force research institute. Due to Van Hooff's involvement a group of chimpanzees was added to Royal Burgers' Zoo in 1971. This latter group was intensively researched by Frans de Waal, van Hooff's first PhD student.
A video published in May 2016 showing the emotional reunion of Van Hooff with Mama, a 59-year-old chimpanzee at the Royal Burgers' Zoo with terminal illness, attracted over ten million views. Mama was the oldest chimpanzee in the Netherlands and had known Van Hooff since 1972. Upon recognising Van Hooff, Mama broke into a wide grin and embraced him. Van Hooff was able to feed and comfort Mama, who had previously refused food. Mama died a week after their reunion.