Science teachers are the primary agents in developing students' scientific literacy. Therefore, it is essential to identify teachers' perceptions of scientific literacy in order to increase students' scientific literacy. This study was designed to develop and validate an instrument of Indonesian science teachers' perceptions of scientific literacy. The questionnaire was developed based on previous research findings and focus group discussions involving six experts. The factors and internal consistency were examined by involving 808 students from various universities in Indonesia. Validity and reliability were tested using exploratory factor analysis and confirmatory factor analysis. This study results showed 41 questionnaire items with acceptable internal validity and consistency. The questionnaires are recommended as a measure of the development of perceptions of prospective science teachers.