The Yorkshire Air Ambulance has received approval from the Civil Aviation Authority to operate at night.
The charity said its two Airbus H145 helicopters would now be able to respond to incidents from 6am to midnight on weekdays and to 10pm on weekends following a successful inspection.
Crews will use night-vision goggles during evening flights and the charity said extending its hours of operation would be of benefit to the five million people it serves.
Captain Andy Lister, director of aviation, said: "This will mean our crew will be operational for longer periods each day, enabling them to respond to incidents into the hours of darkness.
"This will be particularly beneficial in the winter months, when the nights come in much quicker."
The YAA carries out more than 1, 250 missions a year. It's helicopters are housed at bases near Wakefield and Thirsk.