The 40-seat "Bio-Bus", which runs on biomethane gas generated from sewage and food waste, was trialled at weekends earlier in the year.
The gas generated from the annual waste of about five people can power the bus for up to 300km (186 miles).
Operator First West of England will initially run a service between Cribbs Causeway and Stockwood.
To mark the launch, those people who could prove they lived within 400m of the route were offered a free day of travel on the bus.
The biomethane gas is generated at Bristol sewage treatment works in Avonmouth, which is run by GENeco, a subsidiary of Wessex Water.
James Freeman, of First West of England, said: "We're delighted to have the BioBus in service, and hope that as many people as possible try it out for themselves.
"We're really optimistic that this form of gas propulsion can play a significant part in meeting the future transport needs of our area."
A total of 17 million cubic metres of biomethane, enough to power 8,300 homes, is generated annually at the plant through a process known as anaerobic digestion.
The UK's first bus powered entirely by human and food waste has gone into full service in Bristol after testing.