The railway was opened just in time for the Club World Cup football tournament that kicks off Wednesday, with organisers hoping the metro will ease the burden on the wealthy Gulf emirate's choked roads.
The three lines link several stadia, residential areas, the airport and tourist zones.
"We were waiting for this," said airport handling agent Anshu, as she tested the new link between Doha's Hamad airport and the city.
"Almost everybody is taking public transport," she said of her colleagues at the airport, which had previously only been served by expensive taxis and infrequent buses.
A single journey in the metro's standard class, equipped with WiFi and USB charging points, costs two riyals ($0.55), while gold class, boasting plush armchairs and hardwood floors, costs 10 riyals ($2.75).
"For now, its mostly airport staff who are interested. But if people know about it, they will come," said metro assistant Regine.
Construction on the project began in 2013 and a 13 station preview service was launched in May this year.
The network was further tested during the Arabian Gulf Cup football tournament, which concluded on Sunday, with thousands of spectators enjoying free travel to and from stadia for that event.
More than 50 kilometres (30 miles) of tunnels were dug for the first phase of the ambitious project, which has seen 36 stations open to the public.
With high vaulted roofs inspired by traditional Bedouin tents, the stations "reflect the heritage of the country", according to Qatar Rail.
The second phase will see the opening of a new line, as well as the extension of the initial three routes, expanding the network to almost 100 stations by 2026.
In 2022, more than 1.5 million visitors are expected to travel to Qatar, which says its trains have a capacity of one million passengers.
"What is happening now in Qatar is like a movie in fast forward. It's probably a world record between the decision to create a metro system to its opening," chief executive of France's SNCF rail operator, Guillaume Pepy, told AFP.
SNCF runs the service through its majority-owned Keolis unit in a joint venture with a Qatari firm and RATP Dev.
Japan's Kinki Sharyo built the initial 75 three-car trains, split into standard, gold, and family class, and will supply 35 more.
The cost of the project has not been made public.
Qatar has previously said it spends $500 million a week on projects related to the World Cup.