Speaking at Beijing's Great Hall of the People on Saturday, Xi said the Chinese people have a "glorious tradition" of opposing separatism.
"Taiwan independence separatism is the biggest obstacle to achieving the reunification of the motherland, and the most serious hidden danger to national rejuvenation," he said on the anniversary of the revolution that overthrew the last imperial dynasty in 1911.
Peaceful "reunification" best met the overall interests of the Taiwanese people, but China would protect its sovereignty and unity, he said.
"No one should underestimate the Chinese people's staunch determination, firm will, and strong ability to defend national sovereignty and territorial integrity," Xi said.
"The historical task of the complete reunification of the motherland must be fulfilled, and will definitely be fulfilled."
He struck a slightly softer than in July, his last major speech mentioning Taiwan, in which he vowed to "smash" any attempts at formal independence. In 2019, he directly threatened to use force to bring the island under Beijing's control.
China's air force mounted four straight days of incursions into Taiwan's air defence identification zone from 1 October, involving close to 150 aircraft, though those missions have since ended. Xi made no mention of those flights.
Taiwan says it is an independent country called the Republic of China, its formal name. The Republic of China was established in 1912 and its government fled to Taiwan in 1949 after losing a civil war with the Communists, who set up today's People's Republic of China.
Speaking shortly before Xi, Taiwan Premier Su Tseng-chang noted China had been "flexing its muscles" and causing regional tensions.
"This is why countries that believe in freedom, democracy, and human rights, and based on shared values, are all working together and have repeatedly warned that China should not invade Taiwan."
Taiwan marks 10 October, when the anti-imperial revolution began in China, as its national day, and President Tsai Ing-wen will give a keynote address in Taipei on Sunday.