Leaving mosques after prayers in Gaza City, they chanted slogans pledging to defend the mosque and waved green flags, the colour of the Islamist movement Hamas that is de facto ruler of Gaza.
The rally was also attended by other Palestinian factions, including the militant Islamic Jihad.
Ismail Radwan, a Hamas leader, called on "our people in Jerusalem and the West Bank to defend Al-Aqsa."
Israeli police spokeswoman Luba Samri said 5,000 people participated in Friday's prayers at Al-Aqsa, which passed without event.
On Wednesday, four Palestinians were arrested following clashes with police at the Old City site.
Two days earlier, demonstrators clashed with security officers when non-Muslims were to visit the site.
The site is the scene of frequent tensions and also houses the Dome of the Rock, an Islamic shrine.
It is revered by Jews, who call it the Temple Mount, as the location of the biblical Jewish temples and considered Judaism's holiest place.
Non-Muslim visits to Al-Aqsa complex are permitted and regulated by police, but Jews are not allowed to pray at the site for fear it could trigger major disturbances.
Muslims fear Jewish presence on Al-Aqsa is aimed at usurping the site.
On Monday Israeli Prime Minister Benjamin Netanyahu reiterated that "Israel is committed to maintaining the status quo" there.
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