Sulaiman Abu Ghaith entered the plea through a lawyer to one count of conspiracy to kill Americans after being captured in Jordan in the past week.
The US justice department said Abu Ghaith was the spokesman for al-Qaeda, working alongside Bin Laden and current leader, Ayman al-Zawahri, since at least May 2001.
Abu Ghaith is a former mosque preacher and teacher who urged followers to swear allegiance to bin Laden, prosecutors claimed.
The day after the 11 September 2001 attacks, prosecutors say he appeared with Bin Laden and al-Zawahri and called on the “nation of Islam” to battle against Jews, Christians and Americans.
A “great army is gathering against you”, Abu Ghaith allegedly said on 12 September 2001, according to US prosecutors.
Kuwait stripped him of his citizenship after the 2001 attacks. In 2002, under pressure as the US military and CIA searched for Bin Laden, prosecutors said Abu Ghaith was smuggled into Iran from Afghanistan.
Tom Lynch, a research fellow at the National Defence University in Washington, described Abu Ghaith as one of a small handful of senior al-Qaeda leaders “capable of getting the old band back together for a round of international terror”.
The case marks a legal victory for president Barack Obama’s administration, which has long sought to charge alleged senior al-Qaeda suspects in US federal courts instead of holding them at Guantanamo Bay, Cuba. In 2009 he pledged to close the detention centre at Guantanamo Bay. It remains open.