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It will cost £2m a year to run and will be dependent on public donations.
There is a landing base at the Maze Long Kesh site in County Antrim and a back-up at St Angelo outside Enniskillen.
The service will operate with a doctor and paramedic on board. Northern Ireland was previously the only area in the UK without such a service.
The helicopter will be able to reach any part of Northern Ireland within 25 minutes.
"I can't tell you the amount of time it will save in terms of getting a doctor to treat a casualty on site," said Air Ambulance Northern Ireland's Patrick Minne.
"Our first case casualty was taken to Belfast in an eight-minute flight that would probably have taken between 50 and 60 minutes by road."
In that incident last month, Conor McMullan, 11, was involved in a tractor accident in Castlewellan. The air ambulance was scrambled from training to take him to hospital.
The creation of the Helicopter Emergency Medical Service (HEMS) follows a 12-year campaign; the announcement earlier this year that the service would be doctor-led followed an intervention by emergency medics.
Last year, a group of doctors from around the world wrote to then health minister Michelle O'Neill to voice concerns the helicopter service would not have a doctor on board when it was launched.
They were concerned that it could initially be staffed only by paramedics.