Speaking at his first public function since rumors surfaced in early December that his health was deteriorating, Khamenei said Washington could not be trusted - a remark indicating a wary attitude towards President Donald Trump's successor.
In a meeting with organizers of events to mark the first anniversary of the killing of military commander General Qasem Soleimani in a US attack in Iraq, Khamenei said American antagonism would not disappear with the end of the Trump administration.
"My firm recommendation is not to trust the enemy," Khamenei said in remarks carried by state TV.
"The hostility (against Iran) is not just from Trump's America, which supposedly some could say would end when he leaves, as (President Barack) Obama's America also did bad things to the Iranian nation."
Biden was Obama's vice president.
Trump pulled the United States out of the 2015 nuclear deal between Iran and six world powers in 2018 and imposed new sanctions. Biden's coming to power has raised the possibility that Washington could rejoin the agreement.
Some hardline Iranian officials and lawmakers close to Khamenei have questioned President Hassan Rouhani's stand that a revival of the deal may lead to a lifting of sanctions. But Khamenei said he was not opposed to government efforts towards that end.
"If the sanctions can be lifted, we should not delay even one hour...If the sanctions can be lifted in the right, wise... and dignified way, this must be done," he said, addressing government officials.
Earlier, Rouhani said he was happy Trump was leaving office, calling him "the most lawless US president" and a "murderer" for hampering Iran’s access to COVID-19 vaccines.
"We are not overjoyed about Mr. Biden's arrival, but we are happy about Trump leaving … that such a terrorist and murderer, who does not even have mercy for coronavirus vaccines, will be gone," Rouhani said in a televised speech to the cabinet.
Iran says US sanctions are making it difficult for Iran to purchase medicine from abroad, including COVID-19 vaccines needed to contain the worst outbreak in the Middle East.
The Trump administration's sanctions targeted Iran's banking sector and its vital oil industry.
While Washington says medicines and humanitarian goods are exempt from sanctions, the sanctions have deterred some foreign banks from processing Iran's financial transactions.
On Wednesday, Defense Minister Amir Hatami said Tehran had signed arms exports deals with several countries, state media reported. He did name the countries.
TV footage showed Khamenei wearing a mask during Wednesday's meeting and sitting at the head of a large room with several attendees seated to either side, in accordance with COVID-19 social distancing protocols.
The meeting followed rumors on social media earlier this month that the 81-year-old's health was deteriorating.Khamenei has served as supreme leader since 1989, with the final say on all state matters. His health has been the subject of speculation for years.