William will begin a civilian pilot course next month followed by 999-response training. If successful, he will join the East Anglian Air Ambulance based at Cambridge Airport next spring.
The role will be his main job but his roster will take into account his royal duties and responsibilities.
He will be paid a salary that he will donate in full to the charity.
A Kensington Palace spokesman said the Duke, who previously served as an RAF search-and-rescue, pilot was "very much looking forward to" the next step in his career.
Patrick Peal, chief executive of the East Anglian Air Ambulance (EAAA), welcomed the announcement, saying the charity would benefit both from William's skills as a pilot and a boost to its profile.
Mr Peal added: "This is really good news for the charity and we're delighted His Highness has decided to fly with us. We are confident this will help raise the profile of the charity."