Objective To develop a questionnaire assessing Knowledge, Attitudes, and Practices (KAP) Questionnaire on Humanistic Care for Operating Room Nurses and to evaluate its reliability and validity, thereby providing an effective instrument for comprehensively assessing their level of humanistic care-related knowledge, attitudes, and practices. Methods Guided by the Knowledge-Attitude-Practice (KAP) theory, the questionnaire was developed through literature review, Delphi expert consultation, and group discussions. Using a convenience sampling method, 670 operating room nurses from four tertiary Grade A hospitals in Hubei Province were recruited to evaluate the psychometric properties of the questionnaire. Results The final questionnaire consisted of 58 items across three dimensions:knowledge, attitudes, and practices. The scale-level content validity index (S-CVI/Ave) was 0.896. Exploratory factor analysis identified a factor structure with a cumulative variance contribution rate of 63.927%. Confirmatory factor analysis demonstrated an acceptable model fit (χ2/df=1.139, GFI=0.857, AGFI=0.847, NFI=0.828, IFI=0.975, TLI=0.974, CFI=0.975, RMSEA=0.020). The overall Cronbach′s α coefficient was 0.938, and the test-retest reliability coefficient was 0.872. Conclusion The Knowledge, Attitudes, and Practices Questionnaire on Humanistic Care for Operating Room Nurses demonstrated satisfactory reliability and validity and can be used to assess the level of humanistic care-related knowledge, attitudes, and practices among operating room nurses.