According to initial police findings, a white car struck several people in Berlin's Tiergarten park on Saturday night before crashing into a tree, police spokesman Florian Nath said.
The driver left the vehicle and fled in an unknown direction, police said.
"We are conducting an intensive search for suspects," Nath said in a video posted on X.
"There are many injured, among whom multiple people have life-threatening injuries," police said.
Police said they did not yet know the motive or the identity of the suspected perpetrator.
"We currently know nothing about any possible motive or the identity of the perpetrator or anything similar," a police spokesman said.
"None of that has yet been established."
Police officers illuminated the area between Potsdamer Platz and the southern side of the Tiergarten tunnel as they searched for evidence.
Police and emergency services remained at the scene, while visitors were asked to leave the area and avoid the wider area.
Berlin Mayor Kai Wegner described the incident as "an attack on our free and open society".
"After a peaceful and colourful CSD, this gathering for a tolerant and peaceful Berlin was attacked in the most brutal way," he said in a statement.
Wegner thanked the emergency services for their swift response and said he was confident police and security authorities would conduct a thorough investigation.
Organisers said the march was intended to send a message of democracy, tolerance and diversity.
Berlin's Senator for Urban Development, Christian Gaebler, cautioned against speculation over the cause of the incident, saying it was too early to determine whether there was any political or criminal motive.
He noted that the incident had occurred in the Tiergarten park rather than at the Pride march itself, making the situation more difficult to assess.
Hundreds of thousands of people had gathered peacefully in the German capital for the annual Christopher Street Day celebrations, one of Europe's largest LGBTQ events, before the apparent attack.
The Berlin Pride parade is one of the largest events for the lesbian, gay, bisexual, trans, intersex and wider queer community in Europe.
The festivities were called off at about 10 pm local time (6am Sunday AEST) following the incident.
Tabloid newspaper Bild cited a witness as saying a white van drove into the crowd at high speed and a man then got out of the vehicle and fled on foot.
with dpa
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