He responded that he would not support her ambitions because the "customs and traditions" of Palestine prevent this, adding that he agreed with the conservative status quo.
The interview came after the Arab Idol's mother hinted that his recent split from his TV presenter fiancée Lina Qishawi was down to her desire to continue working.
Assaf denied this, but the singer's most recent comments regarding gender roles has received mixed responses from his many fans - both men and women - in the Arab World.
"I feel rather disappointed to see a person that entitles himself the right to deprive another of a dream that he himself is living," said one twitter user Yazan Abu Khalaf.
"It is not reasonable to walk separately and ask for unity at the same time. You want to talk about traditions? From personal experience, I used to be an angry kid in the ghettos of Lyd. I wouldn't calm down unless my mom sang to me a Fairuz song. That is the tradition i want to cherish," said Palestinian rap group DAM on their facebook page.
"So, my dear Arab sisters (hawwa), sing as loud as you can, break the borders so we can calm down. Freedom for all or freedom for none!" they concluded.
Gazan journalist Abeer Ayoub alluded to a similar idea saying that "freedom is liberating the mind" although many retorted that this was a 'foreign' concept of freedom, and not needed or wanted in Palestine.
Many agreed that Assaf is not "against women" but respecting the cultures and values of Palestine.
One Facebook user said that he was a "son of Khan Younis", a conservative city in southern Gaza where Assaf spent much of his life.
Others praised the way that Assaf kept true to his promises not to "forgot where he is from". They said his comments that women shouldn't sing as generally representative of most of Gazan society.
One wrote that Assaf's off-the-cuff comments had perpetrated the unfortunate stereotype of inequality between men and women in the Middle East and that prominent artists should generally keep their views to themselves.
Assaf has not been the only prominent performer who rejected his female relatives from performing.
Actor Adel Imam and filmmaker Majid Masri refused to allow their daughters to become actors. Egyptian singer and actor Tamer Hosni also refused to allow his wife to pursue a career in singing.
Some strands within Islamic thought argue that women should be prevented from singing publically to preserve their modesty. However, this is said to focus more on "traditions" rather than something that is religiously sanctioned.
Assaf is commonly thought to be affiliated with Fatah, the movement that dominates power in the West Bank, whereas the Islamic movement Hamas governs where the singer grew up.
Although many Palestinians from all factions supported Assaf's rise to stardom, but he came under criticism in Gaza for performing "unpatriotic" love songs during the Arab Idol competition.
One prominent sharia scholar in Gaza said that voting for the competition was not in-keeping with Islamic law.
A former Hamas minister also said that Assef should not be performing to an audience of such scantily clad women.
When she was a girl, Niveen Assaf - Mohammed's sister - sang in Assaf's first music video, as well as a popular patriotic Palestinian song.