Demonstrators in the southern city of Khan Younis marched following Friday prayers chanting " Death to Israel" and waving banners reading: "(U.S. President George W.) Bush is a war criminal not a peacemaker."
The conference in Annapolis, Maryland, scheduled to begin Nov. 26, is expected to relaunch formal Israeli-Palestinian talks for the first time in seven years.
Palestinian President Mahmoud Abbas, who fired Hamas from government in June following its violent takeover of the seaside strip, will attend the meeting.
Hamas is not invited to Annapolis, and has railed against the conference, saying it takes place while Hamas-ruled Gaza is under an Israeli blockade and an international boycott, and the meeting therefore does not give a voice to all Palestinians.
Hamas leaders told demonstrators Friday that over the next few days they will hold rallies and public events against the conference, culminating in a Gaza City public meeting to coincide with the Annapolis parley.
"This is the first referendum against Annapolis," said Hamas official Khalil al-Haya. "The world must read what these rallies and conferences mean."
Riham Abu Khater, 17, said she opposed participation at Annapolis as it amounted to recognition of Israel.
"Nothing good will come out of it. Good will only come from the language of fighting, and from force, " she said.
In the north Gaza town of Jabaliya, about two thousand Islamic Jihad activists and supporters took to the streets in protest at Arab participation in the Maryland meeting.
"We consider any Arab effort to make this summit a success as capitulation," said Khaled al-Batch, an Islamic Jihad leader. "We don't recognize any results of this meeting...our response is resistance."