The area around the explosion in Divadelni St was sealed off by police. At least 35 people were injured by the blast, emergency services say, one of them seriously.
Prague's mayor told Czech radio three people may be trapped in the rubble.
Police say that the blast, which blew the windows out of nearby buildings, was most likely caused by a gas leak.
A strong smell of gas was reported before and after the blast by several people in the area, in Prague's Old Town.
The site of the explosion is close to the Vltava river, and near the country's National Theatre.
Neighbouring buildings were evacuated and a two-kilometre stretch of the embankment was cordoned off, Czech media say.
A police spokesman said that there had been about 15 people in the building, which included an office of the International Air Transport Association (IATA) and an art gallery, Reuters news agency reports.
The building also housed a film school and the social sciences faculty of the city's Charles University.
"We heard a strong explosion and we felt the building tremble and the windows shake," eyewitness Jaroslav Faltus said.
Source: BBC News
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