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BACKGROUND Currently, there is no instrument available to assess intensive care unit (ICU) nurses' knowledge, attitudes, and practices (KAP) of central line-associated bloodstream infection (CLABSI) prevention practices. PURPOSE To develop and validate a CLABSI questionnaire to measure ICU nurses' KAP (CLABSI-KAP-Q). METHODS Data were collected from 255 nurses at 4 hospitals in Gansu Province, China. Questions on the CLABSI-KAP-Q were generated through a review of the literature, interviews with nurses, and multiple rounds of content validity evaluation by experts. The validity and reliability of the CLABSI-KAP-Q were assessed with exploratory factor analysis, confirmatory factor analysis, internal consistency, and correlation coefficients. RESULTS The final version of the CLABSI-KAP-Q consisted of 32 items. The reliability was represented by a Cronbach α of 0.946, while the test-retest reliability was 0.945. The overall content validity was 0.95. CONCLUSIONS The CLABSI-KAP-Q is shown to be valid and reliable and recommended for use in clinical practice.