Prince William flew to an incident in Garboldisham in Norfolk on Tuesday. "Patient confidentiality" prevented the EAAA giving details of the mission.
The duke's first shift began at Cambridge Airport on Monday morning.
Later that day what would have been his first active mission to a road accident in St Albans was stood down, EAAA said.
Prince William completed a civilian pilot course in September before taking dedicated 999-response training in order to take up his role with the air ambulance service.
The former RAF helicopter pilot's duties will cover incidents in Norfolk, Suffolk, Cambridgeshire and Bedfordshire.
On Monday the prince admitted to "feeling the nerves" as he began his first nine-and-a-half hour shift as co-pilot alongside medical staff.
However, within three hours he was called on to fly a helicopter to an incident in Hertfordshire, as reported in the Cambridge News.
An EAAA spokeswoman confirmed to the BBC the prince and the rest of the crew were called back to base when it was realised the air ambulance was not required.
Now it has emerged Prince William piloted his first EAAA rescue mission the next day, landing his helicopter in Garboldisham.
"We are unable to confirm any more details of this incident because of patient confidentiality," an EAAA spokeswoman said.
Prince William's shift pattern is expected to be four days on, four days off, but will take into account his official royal duties.
Bond Air Services operates the air ambulance on behalf of the charity and the prince will be paid a salary which he is donating to charity.
The Duke of Cambridge has flown his first active rescue mission as a co-pilot with the East Anglian Air Ambulance (EAAA).