Evidence Based Medicine Questionnaire (EBMQ) was developed to assess the knowledge, practice and barriers towards the implementation of Evidence-Based Medicine (EBM). This study aimed to translate, cross-culturally adapt and psychometrically validate the Persian version of EBMQ. This was an analytical cross sectional study. The EBMQ underwent translation and cross-cultural adaptation following best practices. Face validity was assessed by a panel of five experts. The content validity index (CVI) and content validity ratio (CVR) were evaluated by 15 experts who were familiar with EBM. The tool's internal consistency and test-retest reliability over a 2-week period were evaluated using Cronbach's α and intra-class correlation (ICC), respectively. To assess construct validity, the questionnaire was completed by 400 medical students. Exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) were used for construct validity assessment. All analyses were carried out using IBM SPSS v.24 and AMOS v.24. A significance level of 5% was considered. The CVR for 40 items was higher than 0.62, and their CVI score was 1.0, indicating good content validity. The scale demonstrated acceptable internal consistency and test-retest reliability (n = 30) with an ICC of 0.909 (95% CI: 0.866 to 0.941), and an overall alpha coefficient of 0.957. The structural validity was established through exploratory factor analysis (Bartlett's test p<0.001; the Kaiser-Meyer-Olkin index = 0.862), and it was further confirmed by confirmatory factor analysis. A three-factor solution with 40 items, explaining 51.610% of the variance, exhibited the best fit indices Chi-square statistics/df = 4.23; RMSEA = 0.08; CFI = 0.95; NFI = 0.93; TLI = 0.92). The Persian version of the EBMQ was a reliable and valid tool that could be utilized to assess the knowledge, practice and barriers of EBM for physicians in Persian language countries.