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From Web 1.0 to Web 2.0, the development of Internet technology makes the existing value of Web sites has revolutionary change. How to provide suitable service platform for human-machine interaction through Websites is the subject of debate that many technology developers and users concern about nowadays. This study uses Unified Theory of Acceptance and Use of Technology (UTAUT) as the foundation, from the perspective of tourism companies and Internet users to analyze the relevant factors of Website usability. And then, simple random sampling method would be used for questionnaire investigation, so as to discuss the difference of the application of Website usability towards tourism e-commerce Website service content and the influence towards users' acceptability. The theoretical framework of strategy and service that is applicable to the development of tourism e-commerce Websites are generalized and analyzed, so as to derive out the key indicators of Website usability. The result of this study finds out the users of tourism e-commerce Websites think that "navigation", "functionality and user control", "architectural and visual clarity", "language and content", "consistency", "system and user feedback", "online help and user guides", and "Website obstacle" can promote "behavioral intention" of tourism e-commerce Websites; it also thinks that "error prevention and correction" will promote the "use behavior" of the tourism e-commerce websites. The result of this study can help the tourism companies to view the frameworks of current Websites so as to provide more appropriate services for the users.