ISI leader Sheikh Abu Bakr al-Baghdadi had "made a mistake" by announcing a merger between the two radical Islamist groups in the neighbouring Arab states"without consulting us," he said.
The merger plan has been "damaging to all jihadists", Zahawari said, adding that "Al-Nusra Front is an independent branch of Al-Qaeda".
Zawahiri ordered ISI and Al-Nusra to "cease all hostilities towards each other" and to help each other "in terms of men, money and weapons".
The authenticity of the letter which Al-Jazeera said it obtained from "reliable sources in Syria" could not be independently verified.
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In early April, Baghdadi said that Al-Nusra Front was ISI's branch in Syria and the ISI would be renamed the Islamic State in Iraq and the Levant.
Al-Nusra leader Abu Muhammad al-Julani acknowledged the relationship but rejected the merger and criticised Baghdadi for his announcement.
The declaration caused a stir among jihadists fighting the regime of Syrian President Bashar al-Assad, leading Julani to publicly pledge his allegiance to Zawahiri.
Al-Nusra Front, created in January 2012, joined Al-Qaeda last December on a US list of foreign terrorist organisations. It has carried out some of the deadliest attacks in the uprising against the Syrian regime, claiming responsibility for several suicide bombings.