Background: Evidence-based practice (EBP) education or training are considered fundamental to building and strengthening an EBP culture, as well as to encouraging evidence-based academic and clinical practice in the nursing community. However, few valid and reliable instruments are available for the assessment of EBP teaching and learning in clinical nurses in China. Translation, reliability, and validity testing of the English Evidence-Based Practice Profile Questionnaire (EBP 2 Q), which has strong psychometric properties, may encourage evaluation and promote the implementation of EBP in Mainland China. Methods: Based on established guidelines for the development of questionnaires, the English EBP 2 Q was translated and cross-culturally adapted. The Chinese version of the EBP 2 Q (EBP 2 Q-C) was validated using a sample of 543 nurses. Structural validity was evaluated through exploratory factor analysis and confirmatory factor analysis, and the questionnaire was tested for convergent and criterion validity. The internal consistency and test-retest reliability were also evaluated. Results: The content validity index demonstrated good content validity (≥0.98). An eight-factor structure was obtained in the exploratory factor analysis, and verified by a three-order factor model from the confirmatory factor analysis (χ 2 /df = 2.001; RMSEA = 0.065; SRMR = 0.077; and CFI = 0.884). The Spearman's rank correlation analysis of the EBP 2 Q-C with the Evidence-Based Practice Questionnaire showed moderate correlations for Practice (0.58) and Confidence (0.68) and a low correlation for Sympathy (0.32). Criterion validity was demonstrated by significant differences in terms of nurses' highest education, present position, EBP training, involvement in research programs, and level of understanding of English. Both the overall Cronbach's α and the Cronbach's α for the domains exceeded 0.70. The intraclass correlation coefficients for the domains ranged between 0.75 and 0.96, indicating satisfactory repeatability. Conclusions: Except for the convergent validity of the Sympathy domain, the EBP 2 Q-C provided evidence of validity and reliability. Therefore, it can be applied in EBP education or training assessment in Mainland China.