Khamenei did not appear in person and the remarks were read out by a state television presenter.
No images have been released of him since an Israeli strike at the start of the war that killed much of his family, including his father and wife.
Thursday's statement struck a defiant tone, with Khamenei calling on Iran's neighbours to shut US bases on their territory and warning that Iran would continue to target them.
"I assure everyone that we will not neglect avenging the blood of your martyrs," said the hardline cleric, who is close to Iran's top military force.
"The popular demand is to continue our effective defence and make the enemy regret it. The lever of blocking the Strait of Hormuz must continue to be used," Khamenei added, referring to the shipping route through which a fifth of global oil normally passes along Iran's coast.
State television offered no explanation for why the message was read out rather than delivered in person.
Iranian officials have said Khamenei was lightly wounded in the initial February 28 airstrikes but the extent of his injuries is unclear.
The prospect that one of the most severe disruptions ever to hit global energy supplies could drag on sent oil prices surging back above $US100 a barrel, after falling earlier in the week on hopes of a swift end to the conflict.
There was uncertainty about the strait.
Iranian foreign ministry spokesman Esmaeil Baghaei said some ships could still pass through if they co-ordinate with Iran's navy.
"After the current events, generally we cannot return to conditions before February 28," Baghaei said in comments carried by the Mehr news agency, referring to the start date of the war.
On another front in the unpredictable war, Israeli airstrikes hit a building in central Beirut on Thursday, sending thick smoke above the Lebanese capital.
Israel also ordered residents out of another swathe of southern Lebanon, intensifying its offensive against the Iran-aligned Hezbollah group after it fired its biggest volley of rockets into Israel since the start of the war.
So far the war has killed more than 2000 people, including almost 700 in Lebanon.
Undermining US and Israeli claims to have knocked out much of Iran's stock of long-range weapons, more drones were reported flying into Kuwait, Iraq, the United Arab Emirates, Bahrain and Oman.
with AP
