After completing his bachelor's degree at Yale College in 1996, Dufresne earned his PhD in physics at the University of Chicago in 2000 in the laboratory of David Grier. In the fall of 2000, he and Grier helped to found Arryx, a company that develops holographic optical tweezers and applies that technology. In 2001–2002, Dufresne worked in the Washington, DC, office of the consulting firm, McKinsey & Co. He completed a postdoctoral fellowship with David Weitz at Harvard University from 2002 to 2004.