The charity announced its night flying intentions in a ceremony this afternoon at its base in Redhill.
It is expected to be the first service of its kind in the country to operate at night and the announcement comes following almost three years of research and preparation.
Chief executive Adrian Bell said: "We are committed to ensuring that our life-saving service is available to as many people as possible, regardless of the time of day.
"Although we already attend and treat about 1,000 badly injured and very sick patients per year in daytimes, there has long been clear indication that up to a further 500 patients per year could benefit from the time-critical interventions we are able to provide by night."
Plans suggest the helicopter will be able to respond to as many as three emergencies each night, on top of the four to six missions it can carry out during the day.
Adrian Bell, chief executive of Kent, Surrey and Sussex Air Ambulance
The charity operates two helicopters in daylight - with each one manned by a pilot, a specially trained doctor and paramedic - but only one will be able to fly at night.
This is thanks to the purchase of a new machine, an MD902 Explorer, which replaced the aircraft based in Surrey back in June.
The second helicopter is based in Pattenden Lane, Marden, but does not have the ability to fly at night.
The charity hopes to replace it with another new chopper within a year.
Flight crews are able to provide specialist care at the scene such as general anaesthesia, advanced pain relief and even surgical interventions.
Such procedures are normally only available in the resuscitation area of a hospital emergency department.
Despite the charity being established close to 25 years ago, it was not until 2005 that doctors were put on board.
Initially based in Kent, the purchase of a second helicopter in 2007 saw it expand to cover Surrey and Sussex.
It operates 365 days a year and is capable of delivering anywhere in the region within 20 minutes' flying time.
The service relies solely on donations. It costs around £5million each year to stay airborne and receives no funding from the National Lottery.