Introduction: Unsafe blood products cause transfusion-transmissible infections. A good knowledge and perception about blood safety issues is crucial to ensure safe blood supply. The objective is to develop and validate a question- naire about the knowledge and perception among blood donors on blood safety issues. Method: A cross-sectional study was conducted among 130 blood donors who attended the National Blood Centre, Kuala Lumpur in April and May 2018. The questionnaire was developed in the Malay language after extensive literature search. The self-admin- istrated questionnaire consisted 39 items which required around 20 minutes to complete. The validation involved content validity, construct validity using exploratory factor analysis and reliability using test-retest analysis in IBM SPSS statistics. The same group of respondents was retested after two weeks using the same questionnaire. Results: Content validity was established through multidisciplinary expert meeting and two content reviewers. The factors loadings of all questionnaires were more than 0.40. Knowledge questions were divided into three domains; percep- tion questions were divided into four domains. The intraclass correlation (ICC) values of the test-retest were more than 0.80 for the three knowledge domains and more than 0.60 for the four perception domains. The third domain of the perception section which consisted two questions had the lowest ICC value of 0.686 (95% CI 0.583-0.767). One of the questions was restructured to improve clarity. Conclusions: The questionnaire on knowledge and perception on blood safety issues has good validity and reliability, with appropriate items which warranted its utilization among blood donors.