He urges the faithful to "monitor the targets, collect the money, bring the equipment, plan accurately, and then - while depending on Allah - storm, seeking martyrdom and paradise".
Al-Zawahiri criticises what he sees as collusion between the US, Israel, Egypt and other Arab states against the Hamas-controlled Gaza Strip and its people.
He urges Muslims to support Palestinians through decisive actions against these states, rather than demonstrating their disapproval by other means.
"Let us strike their interests everywhere, just like they gathered against us from everywhere," al-Zawahiri says.
"Let it be known to them that they will get blood for every dollar they spend in the killing of the Muslims, and for every bullet they fire against us, a volcano will turn back on them."
Al-Zawahiri said Israeli jets were bombing people in Gaza "based on the American decision, the Egyptian siege, and Arab collusion".
"They will never be able to insult and make a mockery out of our Prophet, peace and prayers of Allah upon him," he says.
"They cannot expect to support Israel, then live in peace and enjoyment while the Jews are killing our fugitive and surrounded people."
Al-Zawahiri accuses Israel and its allies of restricting Palestinians' access to the most basic necessities and killing them.
The address is the second public message coming from al-Qaeda's leadership in less than a week.
On March 20, Osama bin Laden said in a similar recording that the only way to liberate Palestine is not through negotiations or dialogue, but "iron and fire".
He also urged Muslims to participate in military operations against US forces in Iraq as a means of fighting for Muslim holy places in Jerusalem.
Al-Zawahiri's message also coincides with a visit to the West Bank by Dick Cheney, the US vice-president.
Cheney said on Sunday that attacks on Israel by Palestinian fighters are killing hopes for their "long overdue" state.
Cheney made his first trip as vice-president to the West Bank for talks with Mahmoud Abbas, the Palestinian president, after meetings with Israeli leaders.