Objective:To design an infection control culture assessment scale to comprehensively assess a hospital in general.Methods:Based on the American mature safety culture survey scale(HSOPSC), we developed an infection control culture assessment scale. With this scale, a cross-sectional study was conducted to survey 684 medical staff between October 2018 to January 2019. 607 valid questionnaires were recovered. Reliability and validity of the assessment were then examined by Delphi method, Exploratory factor analysis and confirmatory factor analysis, while the positive response ratio of the dimension was calculated.Results:The scale of infection control culture assessment included 30 entries and 7 factors with eigenvalue>1, and a cumulative variance contribution rate of 57.97%. The 7 factors were basic infection control ability, hospital management atmosphere, mutual support between coworkers, reporting of negative events, interdepartmental collaboration, organizational learning and continuous improvement and working load. Cronbach′s coefficient of the entire assessment was 0.909, and split-half reliability was 0.941. Confirmatory factor analysis showed GFI, CFI, RMSEA, IFI, AGFI and other indicators were better fitted. Among all the factors, the organizational learning and continuous improvement and working load were lower than 50%, which should to be improved for the infection control culture. Conclusions:The assessment scale proved good reliability and validity, which may be widely used in hospitals. Regular measurement and improvement could effectively improve the infection control awareness of medical staff and ensure medical quality.