A total of 27 of the new driver-and-conductor buses, the brainchild of London Mayor Boris Johnson, will be operating on Route 24.
This runs from Hampstead Heath in north west London to Pimlico in central London and is used by 28,000 each day.
A greener, more-accessible successor to the driver-and-conductor Routemaster bus, the New Bus for London is made by the WrightBus company in Northern Ireland.
The first eight of the new buses have already run for some months alongside driver-only vehicles on the route 38 which runs from Clapton and Hackney in east London to Victoria in the centre of the capital.
The next route to get an entire fleet of the new buses will be Route 11 in September this year. This route runs from Liverpool Street station to Fulham Broadway.
In total, 600 of the new buses are on order.
Mr Johnson said: "All the way from Hampstead Heath to Pimlico Londoners can now hop aboard the world's cleanest, greenest bus of its type."
The last regular-service Routemaster ran in December 2005, although some still operate on tourist routes in London in addition to existing services.