The US embassy staff, including marine guards providing security to the embassy, have been transferred to Tunisia “due to the ongoing violence resulting from clashes between Libyan militias”.
Secretary of State John Kerry said there was a “real risk” to staff.
It comes amid fierce clashes between rival militias in the capital, with intense fighting at Tripoli airport.
Libya has been gripped by instability since the 2011 uprising, with swathes of Libya controlled by militias.
The US embassy in Tripoli was already operating on limited staffing. All remaining personnel were driven overland to Tunisia in the early hours of Saturday.
The US military said it had “assisted in the relocation” of embassy staff, using F-16 and MV-22 Osprey aircraft.
It said the five-hour operation was “conducted without incident”.
State department spokeswoman Marie Harf said the withdrawal “underscored the Obama administration’s concern about the heightened risk to American diplomats abroad”.
Marie Harf said that fighting between rival armed groups was taking place “in very close proximity” to the US embassy in the capital.
The state department has also urged US nationals not to go to Libya.
It is the second time in more than three years that the US has closed its embassy in Libya.
Turkey has also withdrawn some 700 members of staff from Libya, Secretary of State John Kerry said.
Earlier this week, the UN also announced it was withdrawing all its staff from Libya.
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