The protest was called by a coalition of Palestinian groups, including Yasser Arafat's Fatah movement and the Islamic radical group Hamas.
The status of Jerusalem is a highly contentious issue, as the city is claimed by both Israel and the Palestinians.
The embassy transfer was included in a bill authorising the US budget for foreign affairs.
Although he signed it on Monday, Mr Bush has said he will ignore the provision about Jerusalem.
The White House released a statement saying the US's policy on Jerusalem had not changed.
Outrage
About 1,500 people took part in Friday's march in Gaza City.
"We condemn the US Congress decision and call on Arabs, Muslims and Christians to intervene for an end to the United States' support for the Israeli aggression," Palestinian member of parliament Ibrahim Abu al-Naja told the crowd.
On Thursday, Mr Arafat also urged "Muslim and Christian nations to act against any decision attacking [the status of] Jerusalem".
On the same day, the Palestinian Authority called for an emergency meeting of Arab ministers to counter the law, while Iran urged the al-Quds (Jerusalem) committee of the Organisation of the Islamic Conference to discuss the issue.
Reversal
The law, which mostly deals with State Department funding, says the transfer of the US embassy to Jerusalem should begin immediately, and stipulates that Jerusalem must be referred to as the Israeli capital in official US documents.
However the White House said this was an unacceptable attempt by Congress to dictate foreign policy, and insisted that US policy on Jerusalem had not changed.
For many years Congress has passed laws urging the US to relocate its embassy to Jerusalem from Tel Aviv - while providing waivers if the president decided against it on grounds of national security.
Successive administrations have used the waiver.
As a candidate, Mr Bush backed Israel's claim to Jerusalem, but in office he has fallen back on the longstanding US position that Arabs and Israelis must settle the question together.
One administration official told the New York Times that the new provision on Jerusalem had proved irresistible to members of both parties in an election year when bipartisan support for Israel was running strong.