The Airbus H135 aircraft, which are fitted with spotlights and carry night-vision goggles, can be deployed for 19 hours a day and until 02:00 GMT.
The Â£5m service will cover Oxfordshire, Buckinghamshire, Berkshire, Hampshire and the Isle of Wight.
It is run by a partnership between the Thames Valley and Hampshire and Isle of Wight air ambulance charities.
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A spokesperson said the decision to end the service at 02:00 was due to the lack of calls relating to major trauma beyond that time, but this could be reviewed in the future.
Previously the air ambulances only operated from 07:00 until 19:00.
Sir Tim Jenner, Thames Valley Air Ambulance chairman, said the partnership between the two charities was "unique".
He said: "Our teams work tirelessly each day and night; not only to deliver a unique life-saving service but to develop their knowledge and training.
"Our ambition of delivering a gold standard service is dependent on the generosity of the people, companies and organisations who support us, to whom we are very grateful."
The service is run in partnership with Bond Air Services Ltd, which provides the helicopters under a long-term contract, and South Central Ambulance Service NHS Foundation Trust.
Two new specially equipped air ambulances have been launched to attend night-time emergencies in the south.