"You [the United States] and your agents are the source of instability in the Middle East... who created Islamic State? America... America's claim of fighting against Islamic State is a lie," the Ayatollah said in a meeting with high-ranking Iranian officials, according to his official website.
He went on to say Iran had no intention of normalising ties with the United States.
"The American government is against an independent Iran... They have problems with the existence of the Islamic Republic of Iran... Most of our problems with them cannot be resolved," the semi-official Fars news agency quoted him as saying.
Iran and the US cut diplomatic ties shortly after Iran's 1979 Islamic revolution and enmity to Washington has long been a rallying point for hardline supporters of Ayatollah Khamenei in Iran.
He has made several statements denouncing the US since the start of the Muslim holy month of Ramadan, while President Donald Trump has spoken out against Iran in harsh terms since taking office, indicating he will reverse the previous admistration's attempts at rapprochement with Tehran.
Mr Trump said Iran must stop funding and training "terrorists and militias" immediately when he gave a speech in Jerusalem last month.
The Iranian leader has also accused the United States and its regional ally Saudi Arabia of funding hardline Sunni militants, including Isis, which carried out its first attack in Iran on Wednesday in Tehran, killing 17 people.
Riyadh has denied involvement in the suicide bombings and gun attacks on Iran's parliament and the mausoleum of the Islamic Republic's founder, Ayatollah Ruhollah Khomeini.
The Iranian President, Hassan Rouhani, who favours opening up to the world, has condemned the attacks without pointing a finger at any country.
The pragmatist president championed a nuclear deal with the US and five other powers in 2015 that led to the lifting of most sanctions against Iran, in return for curbs on its nuclear program.
But the deal has not led to the normalisation of ties between the two countries Mr Rouhani hoped for.
Mr Trump has frequently called the agreement "one of the worst deals ever signed" and said Washington would review it.
Ayatollah Khamenei's hardline loyalists, drawn from among Islamists and the Revolutionary Guards, fear normalisation of ties with the US might weaken their position.
"America is a terrorist country and backs terrorism... therefore, we cannot normalise ties with such country," he said.
Additional reporting by Reuters
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