Objective To develop the Work Fatigue Risk Assessment Questionnaire for Nurses and to test its reliability and validity.Methods Guided by the Accident Causation "2-4" Model, a questionnaire was initially developed through literature review, Delphi and pre-survey.Using convenient sampling method, 513 clinical nurses from a tertiary hospital were selected for questionnaire survey, and the item analysis and reliability, validity test of the questionnaire were carried out.Results The final Work Fatigue Risk Assessment Questionnaire for Nurses included four dimensions of organizational culture, management system, personal ability and personal acts, with a total of 22 items.The cumulative contribution rate was 68.002%, and the confirmatory factor analysis results showed that χ2/df=2.042 （P＜0.001）, RMSEA=0.068,GFI=0.862,CFI=0.933,TLI=0.923,NFI=0.919、RMR=0.021.The correlation coefficients between the scores of each item and the total score of the Fatigue Scale-14 (FS-14) was 0.580-0.667.The total Cronbach′s α coefficient of questionnaire was 0.962, and Cronbach′s α of each dimension was between 0.844-0.945, and the test-retest reliability coefficient was 0.908.Conclusion The Work Fatigue Risk Assessment Questionnaire for Nurses has good reliability and validity, and it can be used as a valid tool for clinical nurses to carry out risk assessment of work fatigue.