The FBI "is investigating the Russian government's efforts to interfere in the 2016 presidential election", Comey told a hearing by the House Intelligence Committee.
"And that includes investigating the nature of any links between individuals associated with the Trump campaign and the Russian government, and whether there was any coordination between the campaign and Russia's efforts," he said.
Comey's disclosure confirmed longstanding reports that the Federal Bureau of Investigation was probing the explosive charges that Trump's stunning election victory over Hillary Clinton last November came on the back of Russian meddling.
US intelligence chiefs said in January they were convinced that Russian President Vladimir Putin was behind that effort.
But they had not commented on whether they were examining links between members of Trump's campaign and Russian officials.
Republican committee chair Devin Nunes opened Monday's hearing - the first public hearing into the issue - by saying the panel had "seen no evidence to date that officials from any campaign conspired with Russian agents".
But Adam Schiff, the Democratic vice chair of the committee, detailed a list of alleged links and communications between the Trump team and Russia.
"Is it possible that all of these events and reports are completely unrelated, and nothing more than an entirely unhappy coincidence? Yes, it is possible," he said.
"But it is also possible, maybe more than possible, that they are not coincidental, not disconnected and not unrelated, and that the Russians used the same techniques to corrupt US persons that they have employed in Europe and elsewhere."
Comey shoots down Trump
The FBI director also shot down President Donald Trump's tweeted claims that his predecessor Barack Obama had wiretapped the phones at Trump Tower in New York.
"With respect to the president's tweets about alleged wiretapping directed at him by the prior administration, I have no information that supports those tweets," Comey told a US congressional intelligence panel.
National Security Agency Director Admiral Mike Rogers also strongly rebutted the suggestion, repeated by Trump's administration, that the NSA had asked the UK's GCHQ intelligence agency to spy on the US president.
"That would be expressly against the construct of the Five Eyes agreement that's been in place for decades," Rogers told the hearing, referring to the intelligence network grouping the US, UK, Australia, Canada and New Zealand.
The combined remarks were perhaps the strongest on-record repudiation yet by senior government officials of the president's extraordinary accusation against Obama.
Comey, speaking to a packed hearing of the House Intelligence Committee, said the lack of wiretapping evidence extended to the department of justice as well.
"The department of justice has asked me to share with you that the answer is the same for the department of justice and all its components," Comey told the panel.
Comey described the "rigorous" US statutory framework, which involves all three branches of government, under which courts grant permission for electronic surveillance.
Asked by the committee's top Democrat Adam Schiff whether Obama could have unilaterally ordered a wiretap of anyone, Comey responded: "No president could."
Trump created a firestorm early this month in a series of accusatory tweets.
"Terrible! Just found out that Obama had my 'wires tapped' in Trump Tower just before the victory. Nothing found. This is McCarthyism!" Trump tweeted.
Last Friday, he repeated his claim, rejecting rising calls from Republicans and Democrats to withdraw the charge and apologise.