The tape, aired for the first time, shows Said al-Ghamidi reading his will and declaring it was a Muslim duty to fight the "American enemy."
"America is the enemy that every Muslim should fight. There is no way the Arab nation can be saved except through jihad," he said.
He vowed that the Mujahidin would stage attacks against US interests.
Ghamidi was in the hijacked United Airlines Flight 93 that crashed in Pennsylvania after passengers on board put up a fight in mid-air.
The tape's broadcast came a day after the world observed the second anniversary of the 9/11 attacks.
An image of Said al-Ghamidi from
the tape broadcast on Aljazeera
The tape is said to have been recorded on 23 December, 2000. It also included a brief audio speech by Usama bin Ladin paying tribute to "the martyr Said al-Ghamidi".
The tape also showed al-Ghamidi firing weapons against the backdrop of what could be Afghanistan's rocky mountains. He was shown jumping out of a truck and firing from an automatic weapon.
Its airing came on a day the CIA authenticated the Usama bin Ladin tape broadcast by Aljazeera a few days earlier.
Broadcast on the eve of the September 11 attacks, the tape showed bin Ladin taking a stroll along an unidentified mountainous slope with his deputy, Ayman al-Zawahri.
The tape scotched earlier speculations that bin Ladin could have been killed in US bombing raids in Afghanistan.