Thursday's flight had been on hold for nearly a year because of COVID-19 border closures and movement restrictions.
Their evacuation followed that of 306 other refugees brought to safety through the so-called Emergency Transit Mechanism set up by Rwanda, UNHCR and the African Union.
The UNHCR-chartered flight, which took off from the Libyan capital, Tripoli, landed safely at Kigali International Airport.
The group included men, women, and children from Eritrea, Sudan and Somalia.
Most were living in Tripoli, but many had previously been held in detention, some for several years.
"I wish governments around the world would help people who are suffering here," said Tsega, an Eritrean asylum seeker.
"The problems we face here are not just being hungry, life in Libya is very difficult, we get kidnapped, they sell us and others buy us.
"Even while we are in the hands of UNHCR we don't feel fully safe because we don't know what we might face at any time while we are here. I want them to help us leave this country as fast as possible."
Asylum-seekers evacuated from Libya are taken to a transit facility in Gashora, where UNHCR is providing them with assistance including medical care, psychological support and language courses.
The group will stay at the transit facility while solutions are sought for them, including resettlement, voluntary return to countries of previous asylum or to countries of origin where it is safe to do so, or integration with local Rwandan communities.
With this latest evacuation, 581 vulnerable refugees and asylum-seekers have been taken out of Libya this year, including 221 via resettlement.
Currently, more than 45,200 refugees and asylum seekers are registered with UNHCR in Libya, including nearly 670 held in government-run detention centres.