Objective To establish an index system evaluating nursing staffs ability in the prevention and control of healthcare-associated infection (HAI), and provide objective standards for their relevant training and evaluation. Methods Evaluation index draft for the ability of nursing staff in preventing and controlling HAI was drawn up based on literature review and semi-structured interview. After screening and inviting 20 experts in the fields of infection prevention and control, nursing management and infectious diseases, draft was revised using Delphi method, and the final index weight was determined with analytic hierarchy process. Results Consultant positive coefficients from two rounds were both 100%, and authoritative coefficients were 0.874 and 0.882 respectively; Kendall's coefficients of concordance fell in the range of 0.139-0.545. Two rounds of expert consultation finally determined the evaluation index system of nursing staff's ability in preventing and controlling HAI, including three first-level indexes, 10 second-level indexes and 28 third-level indexes. Conclusion Establishment of the evaluation index system of nursing staff's ability in preventing and controlling HAI can provide basis for their training and evaluation.