The video, filmed in mid-December, appeared online on Wednesday night and purportedly shows Vanessa Marzullo and Greta Ramelli, who were abducted by gunmen in northern Aleppo province in August.
The women, both in their twenties, are shown wearing black dresses and headscarves, seated before a blank wall.
One woman holds a piece of paper with the date December 17th, though the video appeared to have been posted online on December 31st.
The other, apparently reading from a script held off camera, urges the Italian government to bring them home before Christmas.
The 23-second video includes no details of which group is holding the women and no one else appears in the footage.
The posting on YouTube is titled "Al-Nusra Front detains two Italian employees because of their government's participation in the coalition against it."
But the video was not posted on any official accounts belonging to Al-Nusra, which is Al-Qaeda's Syrian affiliate.
And in previous hostage videos by the group, detainees have been shown in front of Al-Nusra's flag.
In August, the Italian foreign ministry denied reports that the two women were being held by the Islamic State group, but gave no details on which group it believed had kidnapped the pair.
They were working for the aid group Horryaty when they were abducted.
Laura Boldrini, the speaker of the Italian Chamber of Deputies, said authorities in Rome would welcome the apparent evidence that the two women were still alive.
"We had not heard much about them for some time. That they appear to be in good health, even if they have lost weight, is reassuring," Boldrini said.
"I hope that we can bring them home. It hurts to see these young women, full of enthusiasm and altruism, who went there to help others, in this situation."
There was no official comment from the foreign ministry on the video. The country's media gave it high prominence with most newspapers posting it on their websites and broadcasters running still images from it.
Several dozen foreign aid workers and journalists, as well as an Italian Jesuit priest, have been kidnapped in Syria's conflict.
Thousands of Syrians have also gone missing in the war, which began in March 2011 and has claimed more than 200,000 lives.