The sit-in took place at outside Flinders Street Station towards adjoining Swanston Street in the CBD from 5pm on Friday.
Marchers brought Palestine flags and some carried a banner which calls for an end to the war.
Some brought flags or placards, others marched behind a large banner calling for an end to war. (Joel Carrett/AAP PHOTOS)
Organiser Nour Salman said those gathered were calling on Prime Minister Anthony Albanese and his government to take action in response to the conflict.
"We demand a full embargo on Israel across all trade exports and imports," she said.
"We demand implementing immediate sanctions against Israel. The time to act is now.
"Israeli occupation forces have committed and documented hundreds of incidents that break international law."
Pro-Palestinian protesters have staged regular rallies across the nation since October. (Joel Carrett/AAP PHOTOS)
A Victoria Police spokeswoman said it is aware of protest and confirmed officers would be present in the area.
"We respect the right for individuals to protest peacefully, however we will not tolerate those who intentionally block roads and disrupt the lives of the broader community trying to get home or come into the CBD," she said.
More than 30,000 people in the Gaza Strip have died since Israel declared war on Hamas.
The conflict followed Islamic militants crossing the border into southern Israel, killing 1200 people and taking more than 200 hostage on October 7.
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