The new number 38 bus, which will run between Victoria Station and Hackney Central, was delayed for a week because of paperwork.
TfL said a software glitch meant the bus had to be run with its rear platform shut, calling it “teething problems”.
Mayor Boris Johnson said the buses were a “stunning piece of automotive architecture”, which represented the best in British design, engineering and manufacture.
He added that they were “tailored to the London passenger” and that they would help deliver a “cleaner, greener and more pleasant city”.
A “protest” bus – an original routemaster covered in slogans against the rise in London’s public transport fares – followed the new driver-and-conductor vehicle around.
Johnson has been criticised by Labour, the Liberal Democrats and the Green Party because of the cost of the buses.
Eight buses with an open “hop-on, hop-off” platform at the back, costing £11.37 million will run on route 38.
They will be staffed by conductors and won’t run at night or on weekends.
Mike Weston of TfL said he thought the new buses would be “a much-treasured sight on the streets of London”.