The rally, due to begin at midday outside the State Library of Victoria, is being tipped as one of the biggest pro-Palestine protests in Melbourne's history by organisers Free Palestine.
It's the latest of multiple protests held in capital cities across Australia that have been sparked by the war in Israel and Gaza.
Hamas, considered a terrorist organisation by the Australian government, launched an attack on Israel on October 7, killing about 1200 people and taking more than 220 hostages.
In response, Israel has bombarded Gaza, killing more than 12,000 people, 5000 of whom were children.
John Shipton, Mr Assange's father, is expected to address the crowd before they march to Treasury Gardens.
Free Palestine Melbourne representative Muayad Ali said the rally would be the sixth held in the city in as many weeks.
"The Australian public are disgusted at the Albanese government's refusal to call for ceasefire," he said.
"We've had people come who have never before been to a rally and the following week they return with their relatives and friends."
John Shipton, father of Julian Assange, is expected to address the crowd.
On Saturday, the federal government announced fresh sanctions against a number of Hamas members, operatives and financial facilitators, as well as a currency exchange owned by one individual.
The Australia Israel and Jewish Affairs Council said the announcement was a "step in the right direction".
"The war in Gaza is under way because Israel has to urgently remove Hamas' ability to wage war against Israelis and also give Palestinians living under its tyrannical rule in Gaza a chance at a better future," AIJAC executive director Colin Rubenstein said.
Federal Liberal backbencher Julian Leeser, who represents the Sydney seat of Berowra, has urged the NSW government to stop pro-Palestinian road convoys in the state.
He said the vehicle protests created fear in Jewish suburbs and inflamed political and religious tensions.
But Roads Minister John Graham said while Mr Leeser, a Jewish man, was an "outstanding MP" the government did not plan to intervene with people legally driving across the city.
"The focus of the government is on community safety ... we want to make sure in particular that anti-Semitism is not present in this state," he told reporters.
"The government is not keen to restrict movement across the city (but) we are strongly in favour of strengthening the rules around vilification and hate speech."
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