"We reject the cowardly, insolent and vile threats" of Trump against "the sacred sovereignty of Venezuela," Delcy Rodriguez, president of the assembly, wrote on Twitter.
The assembly's 545 members -- all members of the governing party -- would join Maduro "in defense" of the country, she added.
The election of the assembly, decried by the opposition as an attempt by Maduro to install a "communist dictatorship," sharply increased tensions between Caracas and Washington. The two have not had ambassadors in each other's capitals since 2010.
The unexpected warning shocked many Venezuelans. Defense Minister Vladimir Padrino called it "an act of craziness," warning that in the event of US aggression, the nation's military would lead the defense of "the interests and sovereignty of our beloved Venezuela."
On July 31, the United States imposed direct sanctions on the Venezuelan president, terming Maduro a "dictator." To target a sitting head of state is an extremely rare move.