"We do not recognise [...] anyone else's right to dictate to you or any other country what alliance you will seek to belong to or what bilateral relationships you have," he said in an apparent reference to Russia's influence on Ukraine, which wants to join NATO.
Biden made his comments at a joint news conference with President Viktor Yushchenko during a visit to the ex-Soviet country, seen as a move to balance US President Barack Obama's visit to Moscow earlier this month.
Biden's national security advisor, Tony Blinken, told reporters that Biden stressed Ukraine still had far to go before it could join the Western military alliance.
Obama's commitment to improving ties with Russia after a period of poor relations "will not come at Ukraine's expense," Biden said. "To the contrary, I believe it can actually benefit Ukraine."
Yushchenko, who came to power after a pro-Western revolution in 2004 in which he benefited from US support, said US-Ukrainian relations should be developed in a "constructive way".
"We don't want to see them [made] at the expense of Ukraine or at the expense of the creation of any zones of special interests," he said.
Closer ties with West
Yushchenko has sought to drive his country toward closer ties with the West, including membership of the US-led military alliance NATO, an aspiration which has particularly annoyed Russia.
He restated his aspiration to move Ukraine closer to NATO.
"We believe that the best way to respond to the politics of security, as we have outlined in our law, is to develop European-Atlantic dialogue," he said.
Under pressure from Moscow, with which it is trying to improve relations, NATO has held back from fast-tracking Ukraine to membership, though it has said it will be a member one day.
"The vice-president [told Yushchenko] that NATO membership is not only a right," Blinken said.
"There will be a lot of work to get Ukraine to a place where it is ready for membership. We want to do that work, if that is what Ukraine wants, and NATO wants to do that work too."
Yushchenko's term finishes early next year and polls show he has almost no chance of being re-elected in a 17 January election.
The leading contenders to succeed him, who will also meet Biden, generally favour a less confrontational approach toward Russia, with which many Ukrainians share a common language.
Russian Foreign Ministry spokesman Andrei Nesterenko said Russia would watch Biden's visit closely.
"The main thing is that this happens transparently without any under-the-carpet games and not at the expense of anyone else's interests," he told reporters in Moscow.
Feuding between Yushchenko and Prime Minister Yulia Tymoshenko, once allies in the 2004 'Orange Revolution', have paralysed decision-making and raised concerns in Washington about Ukraine's stability.
Yushchenko also said Ukraine would like the United States to help finance the modernisation of its gas transit network, which he called "an integral part of the European gas market".
Kiev has angered the Kremlin by asking investors from the European Union to help modernise the network, through which a fifth of Europe's gas needs run from Russia.
(EurActiv with Reuters.)
