In a 22-minute recording, posted on Islamist websites on Thursday to mark 65 years since Israel's founding, Zawahri also said the only way to solve the Palestinian problem was through Jihad, an Islamic holy war.
"Lions of the Levant, unite around this honorable goal and rise above party affiliations," Zawahri said.
"America, its agents and allies want you to shed your blood and the blood of your children and women to bring down the criminal Baathist regime, and then set up a government loyal to them and to safeguard Israel's security," he added.
The recording came one day after Syrian government forces, backed by Lebanese Hezbollah fighters, captured the border town of Qusair from rebels. It appeared on the Mujahideen al-Ansar website, which carries statements from Al-Qaida leaders.
It was not immediately possible to verify the authenticity of the recording, which made no reference to Qusair, suggesting it was recorded before the campaign to retake the town began more than two weeks ago.
Zawahri, an Egyptian-born preacher who became Al-Qaida's chief after Osama bin Laden was killed in a U.S. operation in 2011, also criticized Iran for supporting Assad, saying the conflict in Syria had "revealed the ugly face of Iran."
The Iranian-backed group Hezbollah fought with Assad's forces which seized the strategically important border town of Qusair from rebels on Wednesday.
Influential Sunni Muslim cleric Sheikh Youssef al-Qaradawi last week called for Jihad against Assad after Hezbollah leader Sheikh Hassan Nasrallah acknowledged his forces had joined the fighting in Qusair.
Zawahri warned Syrians not to play into the hands of the United States which, he said, wanted to turn their Jihad into a "Western tool against Iran."
He said that fighters seeking to topple Assad in Syria had rekindled hope that one day that might happen.
"There is no solution for Palestine except Jihad," Zawahri said.
"Every free Muslim in Palestine should unite with his Muslim brothers to implement Sharia (Islamic law) and rule by it, and make it a reference above all references, and to liberate Palestine in order to set up an Islamic state, even if the West hates that and calls it terrorism and extremism," he added.
Zawahri's last audio message dates to April, when he also urged Muslims to unite and support Jihad to create a state governed by Islamic law.
Want to enjoy 'Zen' reading - with no ads and just the article? Subscribe todaySubscribe now