On Thursday, Mulino also rejected the possibility of reducing tolls for American vessels going through the canal.
"There's nothing to talk about," Mulino told a press conference.
"The canal is Panamanian and belongs to Panamanians. There's no possibility of opening any kind of conversation around this reality, which has cost the country blood, sweat and tears," he added.
His comments came after Trump criticized fees for US ships using the canal while complaining about China's growing influence in Panama.
"It was solely for Panama to manage, not China, or anyone else," Trump said in a post on his Truth Social platform. "We would and will NEVER let it fall into the wrong hands!"
If Panama could not ensure "the secure, efficient and reliable operation" of the channel, "then we will demand that the Panama Canal be returned to us, in full, and without question," he said.
Rejecting Trump's allegation, Mulino asserted that there is absolutely no Chinese interference or participation in anything to do with the Panama Canal.
Mulino also denied the allegations of Trump who said on Wednesday that Chinese soldiers were "lovingly, but illegally, operating the Panama Canal."
"There are no Chinese soldiers in the canal, for the love of God," Mulino added.
On Tuesday, dozens of demonstrators gathered outside the US embassy in Panama City, burning an image of the incoming US president.
The canal was built by the United States and inaugurated in 1914, but was handed to Panama on December 31, 1999, under treaties signed between the two sides about two decades earlier.
MNA/