Objective: To develop a questionnaire assessing nursing staff's knowledge, attitude, and practice on the prevention of the nosocomial infection in elderly patients and test its reliability and validity. Methods: After the drafted questionnaire was developed, two rounds of Delphi survey were conducted by consulting experts to improve the questionnaire. Subsequently, 700 copies of the questionnaire were distributed to nursing staff to assess its reliability and validity. Results: Exploratory factor analysis (EFA) identifies 3 aspects, namely knowledge, attitude, and practice, with a total of 38 items. The Cronbach's a coefficients of the questionnaire and each of the aspects are 0.85, 0.80, 0.886, and 0.77 (>0.7), respectively. Confirmatory factor analysis (CFA) of each of the aspects are c2/df = 3.99, 2.26, and 3.32; Goodness-of-fit index (GFI) = 0.91, 0.97, and 0.92; Root mean square error of approximation (RMSEA) = 0.06, 0.04, and 0.05; Comparative fit index (CFI) = 0.91, 0.96, and 0.90. Conclusions: Through this study, it can be ascer tained whether the developed questionnaire enjoys sound reliability and validity in assessing nursing staff's knowledge, attitude, and practice on preventing the nosocomial infection in elderly patients and thus has cer tain application value.