The purpose of this study was to develop the Critical Thinking Disposition Test in Biology as an alternative instrument in looking at the extent of one's disposition to critical thinking, especially in Biology University. Critical Thinking Disposition Tests in Biology are tests in the form of multiple choice based on biological cases. This research was applied at the University in Bengkulu which involved six experts as reviewers and 320 students as respondents, namely with the level of study: year 1, year 2, year 3, and year 4. This research refers to the design of development and validation. Data collection techniques used were content validation formats and questionnaire. The results of expert validation were analyzed by the Content Validity Ratio (CVR) method, while the results of the construct validation were analyzed through Confirmatory Factor Analysis (CFA). The results show that the Critical Thinking Disposition Test in Biology has good content validation and construct validation.