The UN says the aid includes food, winter clothing and blankets as well as hygiene kits.
The refugees, mostly women and children, live in dire conditions in two makeshift camps in a desert area.
Jordan shut the border in June after seven of its soldiers died in a bombing claimed by Islamic State militants.
Since then only a single aid delivery was allowed when food rations were sent using cranes in August to the Rukban and Hadalat camps.
On Tuesday, the UN said it had reached an agreement with the Jordanian military after protracted negotiations.
"The resumption of assistance comes at the start of the coldest period of the year, when temperatures can drop dangerously low," a UN statement said.
The refugees on Jordan's border are said to be among the most desperate of the millions displaced by Syria's bloody war, the BBC's Yolande Knell reports.
There are now also plans to open a clinic in the area, our correspondent adds.
The UN estimates that more than 600,000 Syrian refugees are now in Jordan, but the government in Amman says the number could be as high as nearly 1.5 million.