He was the son of Sir Francis Willughby, and was born at Middleton Hall, Warwickshire. He studied at Sutton Coldfield school and Trinity College, Cambridge.
At Cambridge he was taught by the naturalist John Ray. Between 1663 and 1666 they toured Europe together, and on returning to England made plans to publish the results of their studies. Willughby died during the preparation of this work, but Ray published Willughby's Ornithology in 1676. This is considered the beginning of scientific ornithology in Europe, revolutionizing ornithological taxonomy by organizing species according to their physical characteristics.
The Willughby family seat, Wollaton Hall, now owned by the City of Nottingham, houses Willughby and Ray's natural history collection of stuffed animals and birds.