Businessman Robert Tsao and lawmaker Puma Shen are now barred from entering mainland China or the special administrative regions of Macau and Hong Kong, said Chen Binhua, a spokesperson for Beijing's Taiwan affairs office.
Beijing considers Taiwan part of its own territory and has not ruled out using force to bring it under control.
The Chinese military launched its fourth round of war games targeting Taiwan in just over two years on Monday in what it called a "stern warning" to alleged pro-independence forces on the island.
Tsao's and Shen's family members will face similar bans and any businesses affiliated with them will be barred from "seeking profit" in China.
Chen said Chinese authorities would also block any activity on the mainland by Kuma Academy, a civil-defence training organisation founded by Shen and to which Tsao -- founder of Taiwanese microchip maker United Microelectronics Corporation -- has donated money.
The group aims to prepare ordinary citizens for war and "increase determination to defend and resist invaders, ultimately building a protective shield for our country", according to its website.
Chen accused the two Taiwanese of "nakedly splitting the country, inciting separatism, severely harming the stability of the Taiwan Strait, severely damaging the shared interests of compatriots on both sides of the strait and the fundamental interests of the Chinese nation".
Shen hit out at Beijing's sanctions and told reporters on Monday it was no coincidence they were announced during the drills "since it is China flying the planes, it is clearly China that is actually engaging in provocation".
Tsao called Beijing "an ancient ghostly regime that disregards popular sovereignty" in a Facebook post after the announcement.
Kuma Academy also published a statement on its official Facebook page describing Beijing's accusations as "purely politically motivated, aimed at undermining our mission and silencing voices that support Taiwan's right to self-determination and democratic values".
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