The video carried by state media showed dozens attending a ceremony to mark Ashura, the holiest day of the Shi'ite Muslim calendar, standing chanting as Khamenei entered a hall where many government functions are held.
Khamenei last appeared in public on June 11 at a meeting with members of parliament, two days before Israel launched its attacks on Iran.
For apparent security reasons, Khamenei had issued pre-taped messages during the war which started on June 13 and avoided public appearances.
In a country that relies on symbolic presence and religious authority, Khamenei's absence was notable and gave rise to intense speculation over his whereabouts.
The supreme leader was openly threatened by Israeli Defence Minister Israel Katz during the conflict.
Iranian authorities have acknowledged the deaths of more than 900 people in the air war with Israel, as well as thousands of injured.
It also has confirmed serious damage to its nuclear facilities, and has denied access to the sites for inspectors with the United Nations nuclear watchdog.
Israel relentlessly attacked Iran beginning June 13, targeting its nuclear sites, defence systems, high-ranking military officials and nuclear scientists.
In retaliation, Iran fired more than 550 ballistic missiles at Israel, most of which were intercepted, but those that got through caused damage in many areas and killed 28 people.
with DPA and AP
