"The import of American and British vaccines is prohibited", Khamenei said in his address on Friday.
Mr Khamenei praised Iran's efforts to develop domestic vaccines but said Iran could obtain vaccines "from other reliable places".
Iran has been the worst-hit country by the coronavirus in the Middle East.
Where will Iran get its vaccines?
Even though food and medicine are exempt from these sanctions, worldwide banks tend to decline transactions that involve Iran.
Iran's President Hassan Rouhani said last month that Washington had demanded that Tehran pay for the drugs through US banks, adding that he had feared the United States would seize the money.
Mr. Khamenei's edict bans imports of the Pfizer-BioNTech, Moderna and AstraZeneca-Oxford anti-Covid 19 jabs. Iran's Revolutionary Guard in December rejected the use of foreign-made vaccines altogether. The Trump administration imposed crippling sanctions on Iran while demanding it renegotiate stricter terms to the deal.
Iran-backed militias besieged the U.S. Embassy in Baghdad late past year, sparking a series of events that culminated in the Soleimani assassination.
Iran launched human trials of its first domestic COVID-19 vaccine candidate late last month, saying it could help Iran defeat the pandemic despite US sanctions that affect its ability to import vaccines.
The Red Crescent in Iran said it will not import the massive stash of the Pfizer vaccine that benefactors from America had planned to donate to the country.
U.S. President-elect Joe Biden has said the United States would return to the JCPOA if Iran returns to compliance, followed by further negotiations addressing additional issues such as the regime's ballistic-missile program.
The German BioNTech vaccine is co-produced by the American firm Pfizer, the Moderna vaccine is a U.S. achievement and the Oxford University-AstraZeneca vaccine - cheaper than the other two options - is from the UK. In those cases, prosecutors said people were infected when they received transfusions of HIV-tainted blood that came from France. Soleimani led Iran's terrorism efforts in the Middle East and has been responsible for the deaths of hundreds of American soldiers.
France's then-prime minister Laurent Fabius was charged with manslaughter, but acquitted in 1999, while his health minister was convicted but never punished.