Al-Nusra Front leader Abu Mohammed al-Jawlani formally confirmed on Wednesday that his rebel group was allied to al-Zawahiri in an audio recording posted online.
"The sons of al-Nusra Front renew their pledge to the Sheikh of Jihad Ayman al-Zawahiri and declare obedience," al-Jawlani said.
The move is likely to fan fears that radical Islamists are gaining influence in Syria and bolster assertions from President Bashar al-Assad that the regime is fighting "terrorists."
However al-Jawlani sought to distance his group from claims made a day earlier by al Qaeda's Iraqi branch that al-Nusra Front had merged with the organization.
"We inform you that neither the al-Nusra command nor its consultative council, nor its general manager were aware of this announcement. It reached them via the media and if the speech is authentic, we were not consulted," Jawlani said, referring to the announcement by al Qaeda in Iraq chief Abu Bakr al-Baghdadi.
He added that despite the allegiance, the conduct of the group would remain unaltered.
"We reassure our brothers in Syria that al-Nusra Front's behavior will remain faithful to the image you have come to know, and that our allegiance will not affect our politics in any way."
West considers support for rebels
Long-held suspicions of links between the al-Nusra Front and al Qaeda have prompted the United States to label the group as a terrorist organization.
As one of the most prominent rebel groups in northern and eastern Syria, it has become known for employing suicide bombings in the fight against the regime.
Wednesday's announcement is likely to complicate western attempts to arm the Syrian opposition.
The US and EU currently provide non-lethal aid to the rebel movement. Britain and France have appealed to the EU to lift and arms embargo on Syria, however most nations have blocked such a move, fearing weapons could enter into the hands of al Qaeda-linked militants.
According to the United Nations at least 70,000 people have been killed since the uprising against al-Assad's regime began in March 2011.
ccp/msh (AFP, Reuters, AP)