Objective To evaluate the reliability and validity of questionnaire of acquired immunodeficiency syndrome prevention for senior high school students. Methods The questionnaire of acquired immunodeficiency syndrome prevention for senior high school students was designed on the basis of health promotion theory. A total of 12 313 students of grade 1 in senior high schools were selected by cluster random sampling from Shanghai, Sanming of Fujian Province and Beihai of Guangxi Zhuang Autonomous Region, and survey with the questionnaire was conducted. Two hundred and fifty-one students were retested, and the reliability and validity of the questionnaire were examined. Results Cronbach's α coefficient of the reliability of the questionnaire ranged from 0.608 to 0.916. Spearman correlation coefficients were all higher than 0.7 in the evaluation of test-retest reliability. The construct validity of the questionnaire was good by factor analysis, and 15 common factors including knowledge, attitude, belief and practice could explain 58.381% of the questionnaire. Conclusion The questionnaire of acquired immunodeficiency syndrome prevention for senior high school students designed in this study has favorable reliability and validity, and can be applied in related researches.