Scottish airline Loganair will operate the frequent service to London Stansted from March 30.
It will replace the existing CityJet service to London City which is due to be withdrawn at the end of that month, officials said.
Transport minister Keith Brown hailed the announcement as "welcome news" for Dundee Airport and said it guarantees continuity of service for the Dundee-London route.
London Stansted, a major hub for low-cost airlines, is the fourth busiest airport in the UK and it serves around 140 destinations in 30 countries across the UK, Europe, North Africa and Asia.
Loganair will operate a twice-daily service every day of the working week and once during the weekend.
The weekday flights will leave Dundee at 7am and 4.25pm, with return flights departing Stansted at 9am and 6.25pm.
There will also be a flight to and from London every Sunday afternoon, leaving Dundee at 4.25pm and departing Stansted at 6.25pm.
The new service, operated by a 32-seat Dornier 328 turboprop aircraft, is said to be the result of weeks of negotiation between Dundee Airport operator HIAL, Dundee City Council, Transport Scotland and the airline.
In the meantime, tenders will soon be issued to secure a public service obligation that will ensure the long-term future of air services between Dundee and London, officials said.
HIAL managing director Inglis Lyon said: "Stansted is a great choice of airport for Dundee, with a wider choice of airlines and three times the number of destinations on offer at London City.
"Flight times have been designed with business passengers in mind and will allow those travelling for work to reach the city in good time for an early-morning meeting, with easy rail access into Liverpool Street, right in the heart of London's financial centre.
"Leisure passengers will also enjoy a greater choice of onward connecting airlines and destinations while those heading for a London city break will find Stansted well-placed for the city's top attractions."
Mr Brown said: "Maintaining this link is crucial to the airport's long-term future and this announcement safeguards the service as we move towards establishing a full public service obligation on the route.
"Transport Scotland continues to work hard in partnership with Dundee City Council and HIAL to find ways of maximising the potential of Dundee Airport."
Flights can be booked from Tuesday via Loganair's franchise partner Flybe at www.flybe.com, or by calling 0871 700 2000. Fares start from £74.99 one way including tax and include a free 20kg bag.