The deadly violence in the city's historic center sparked evacuations and a massive response by heavily armed police, including warnings for people to stay indoors.
The incident occurred at the Charles University's Faculty of Arts, which sits near major tourist sites like the 14th-century Charles Bridge.
"More than 15 people have lost their lives and at least 24 have been wounded," police chief Martin Vondrasek told reporters following the shooting.
Emergency services reported nine serious injuries, at least five mid-serious and up to 10 light injuries.
Vondrasek said police started a search for the man before the mass shooting as his father had been found dead in the village of Hostoun west of Prague.
An ambulance drives towards the building housing the Philosophical Faculty of Charles University in downtown Prague, Czech Republic on Thursday, December 21, 2023.
The gunman "left for Prague saying he wanted to kill himself," Vondrasek said. Police suggested earlier the gunman had killed his father.
Police searched the main Faculty of Arts building where the gunman was expected to show up for a lecture, but he went to the faculty's other building nearby and they did not find him.
"At 13:59 GMT, we received the first information about shooting," Vondrasek told reporters, adding the rapid response unit was on the scene within 12 minutes.
"At 14:20 GMT, the officers in action told us about the gunman's motionless body," Vondrasek said, adding unconfirmed information showed he had killed himself.
Citing a probe into social media, Vondrasek said the gunman was inspired by a "similar case that happened in Russia this autumn," without further details.
"At the moment, there is nothing to suggest any further imminent danger," he added.
The White House on Thursday condemned the "senseless" shooting at the Charles University of Faculty Arts in Prague and said the United States was ready to offer assistance.
"The President (Joe Biden) and the first lady (Jill Biden) are praying for the families who lost loved ones and everyone else who has been affected by the senseless act of violence," White House Press Secretary Karine Jean-Pierre told reporters.