The closure at the end of March had a severe impact on trade between the two countries and left Libyans and Tunisians stranded on either side of the frontier.
On the Libyan side of the border, several cars waited to cross into Tunisia while some people, wheeling luggage, walked to the other side.
Border police checked the identity of the travellers and ensured they were carrying negative tests for Covid-19, AFP correspondents said.
Many Tunisians travel to Libya for work, while Libyans go regularly to Tunisia for medical treatment.
“We will be prioritising the sick and the ones who have special cases,” Abu Rabih Malkhuf, head of security on the Libyan side of the border, told AFP.
“There is a special protocol that will be followed, and protective measures will be taken, such as wearing masks and social distancing,” he added.
Tripoli resident Ahmad Moftah waited impatiently for his turn to enter Tunisia, where said he hoped to receive medical treatment.
“The closure (of the border) was too long,” he added.
Health workers wearing face masks and blue personal protective gear gathered with sanitising equipment.
“Work at the crossing will continue throughout the day. The workers are trained to sanitise the passengers, cars and trucks,” Hassan al-Gomati from Libya’s pandemic task force said.
Tunisia closed its border with Libya in March as part of measures to contain the spread of the novel coronavirus pandemic.
In April, more than 600 Tunisians stuck in the neighbouring North African country made their way back home after arriving at the frontier en masse, with Tunisian border guards letting them through.
A Tunisian diplomatic source said 20,000 Tunisians were currently in Libya.
Dozens of travellers began crossing from Tunisia into Libya when the border opened at midday Saturday, while several trucks transporting merchandise waited their turn, an AFP correspondent said.
Informal trade between the two countries supports thousands of families in southern Tunisia.
Air links are due to resume Sunday, Libyan and Tunisian authorities said.
Tunisia had reopened its borders at the end of June, allowing travel from Europe during the tourist season, but had kept them closed to neighbouring Libya and Algeria.