In a video link, Mr Biden voiced deep concerns over Russian troop build-ups and threatened "strong economic and other measures", Washington said.
Russia says it will not attack Ukraine.
Mr Putin accused Kyiv of provocation, and sought guarantees against eastward Nato expansion and deployment of offensive weapons close to Russia.
The talks began at 10:07 US Eastern time (15:07 GMT; 18:07 Moscow time), the White House said.
They were held on a secure video link set up under previous administrations but never used before, Russian news agency Tass said.
Video footage of the opening moments showed friendly greetings between the US and Russian leaders. The talks then continued behind closed doors, lasting about two hours.
Mr Putin held the talks from his residence in the southern resort of Sochi, according to Tass.After Tuesday's video call, US National Security Adviser Jake Sullivan told journalists Washington was preparing specific robust responses in the weeks ahead if they were required.
"Things we did not do in 2014 we are prepared to do now," he said, referring to Western responses to Russia's annexation of Crimea.
"There was a lot of give and take, there was no finger-wagging, but the president was crystal clear where the United States stands on all of these issues," he added.
The measures included economic sanctions and other actions such as additional troop deployments to Nato allies in the region and defence materiel for Ukraine.
He refused to be drawn on what the economic measures might be, saying the US would prefer to communicate them directly to the Russians.
But he said that Nord Stream 2, a new pipeline to Germany which is not yet in operation, provided "leverage" for the US and its allies.
"If Vladimir Putin wants to see gas flow through that pipeline, he may not want to take the risk of invading Ukraine," he said.
Earlier reports said US officials had reached agreement with Germany to shut down the pipeline in the event of an invasion.
Other possible measures include restrictions on Russia's banks converting roubles into foreign currencies, or even disconnecting Russia from the Swift global financial payment system, reports say.
'Reliable guarantees'
Meanwhile a Kremlin statement (in Russian) said Mr Putin had stressed that Russia should not be held responsible for tensions because Nato was making "dangerous attempts to take over Ukrainian territory and increasing its military potential" on Russia's borders.
"Therefore Russia is seriously interested in getting reliable guarantees fixed in law to rule out the eastward expansion of Nato and the location in countries neighbouring Russia of offensive weapons systems," the statement added.
Ukrainian President Volodymyr Zelensky said that, while the talks brought "no sensations", he was grateful for Mr Biden's "unwavering support".
Mr Biden and Mr Putin last met in person in Switzerland in June, but made little progress other than to agree to send their ambassadors back and begin a dialogue on nuclear arms control.
In a conference call on Monday night, the White House said the leaders of the US, UK, France, Germany and Italy had formed a joint strategy "to impose significant and severe harm on the Russian economy" should Russia launch an invasion.
Mr Biden spoke to the four European leaders again after his talks with Mr Putin.
More than 90,000 Russian troops are believed to be massed near Ukraine's borders.
A large part of the recent Russian military build-up is in Crimea, which Russia seized from Ukraine and then annexed in 2014.
Troops are also gathering near Ukraine's eastern Donbas region, the name for parts of Luhansk and Donetsk regions which are under the control of Russian-backed separatists.
Ukrainian officials have said Moscow could be planning a military offensive at the end of January.
More than 14,000 people have lost their lives in seven years of conflict since Russian-backed forces seized large areas of Ukraine's east.
