The innovation by First Bus and Aberdeen City Council follows the first hydrogen-powered single decker buses being trialled in the city from 2015.
The £500,000 vehicles emit only water, are virtually silent and are said to save 1kg of carbon dioxide per kilometre.
They take around ten minutes to refuel and will operate mainly on the First’s service 19, along with other routes.
The vehicles are powered by a fuel cell at the rear which converts hydrogen into electricity.
The buses carry six hydrogen tanks, four beside the fuel cell and two behind seats at the rear of the top deck.
First Aberdeen engineering manager Michael Smith said: “They are carbon fibre wrapped and completely safe.”
City council co-leader Jenny Laing said: “Aberdeen is one of Europe’s pioneering hydrogen cities and through the work of the council, the city has developed a cluster of hydrogen activity.
"They have even more advanced technology which pushes established hydrogen boundaries and will greatly assist us in tackling air pollution in the city.”
First Bus managing director Andrew Jarvis said: "Scotland will be home to [UN climate change conference] COP26 later this year and it is fantastic to know we'll be setting a stellar example of just what can be achieved with new technology as we welcome leaders from around the world.”
The £8.3 million scheme has been part funded by the European Union’s Jive project to aid the development of hydrogen buses, along with the council and Scottish Government.