At a ceremony at the Jabaliya refugee camp in the north of the devastated Palestinian territory, a spokesman for the Ezzedine al-Qassam Brigades -- the military arm of the Islamist Hamas movement -- said 2,500 recruits would form "the first section of the popular army for the liberation of Al-Aqsa and of Palestine".
Mohammed Abu Askar, a Hamas official, said those older than 20 could sign up "to be prepared for any confrontation" with Israel.
Hamas and Israel fought a 50-day war earlier this year which resulted in the deaths of 2,140 Palestinians and 73 Israelis, and destroyed swathes of the Gaza strip.
Signup to our newsletter and follow us on Facebook and Twitter!
Frequent clashes between Palestinian protesters and Israeli police occur around the Al-Aqsa mosque compound in occupied east Jerusalem.
Recent confrontations have been largely triggered by Palestinian fears that Israel was poised to change the status quo and allow Jewish prayer at the site, which is holy to both Muslims and Jews.
Abu Askar said the new force had been established "at a moment when the Al-Aqsa mosque is subject to serious Israeli violations".
Another Al-Qassam spokesman said "the people, arms and the tunnels are going well", in reference to Hamas's network of underground passages, hundreds of which have been destroyed by Israel and Egypt to prevent arms smuggling.