According to Malian and Swiss authorities, armed men arrived at the home of Beatrice Stockly in a convoy of four vehicles.
Confirming the abduction, army spokesman Souleymane Maiga said: "I confirm that a European woman was kidnapped in Timbuktu at 3.30 a.m. (0330 GMT).
"A neighbour alerted the security forces around 6 a.m."
No one has claimed responsibility for her second abduction but al-Qaeda linked fighters are known to operate in the area.
Beatrice Stockly was taken by Islamic militants in 2012 but was released after four days.
It's claimed that despite French forces driving Islamist fighters out of urban areas back in 2013, attacks have intensified in recent months.
France still has armed forces on the ground in the African country along with the UN, which has a a 10,000 strong peacekeeping force in operation.