Background: Inadequate awareness of livestock breeders concerning the transmission of brucellosis and their improper knowledge with regard to the prevention of brucellosis are among important barriers to vaccinate animals against brucellosis. The aim of this study is design and validation of a Brucellosis prevention questionnaire focused on animal vaccination . This valid questionnaire is used to design, implement and evaluate an interventional training program. Methods: In this exploratory psychometric study, a brucellosis prevention questionnaire (BPQ) was developed. Face-to-face interviews were conducted to develop the initial items of BPQ. The findings from interviews were merged with those of literature review. Face, content and construct validity of BPQ were assessed by participation of livestock breeders, veterinarians and health educationists. Impact Score (IS), Content Validity Ratio (CVR) and Content Validity Index (CVI) of the items were calculated. Construct validity of BPQ was evaluated through factor analysis. Cronbach’s alpha, Intra-class correlation coefficient (ICC), composite reliability and standard error of measurement (SEM) were measured to assess the reliability of the findings by BPQ. Results: BPQ was finalized by 53 items and its validity was confirmed by CVI, CVR, and IS of 0.90, 0.74 and 4.30, respectively. The items were loaded into three constructs of awareness, attitude and practice. The predictive power of the awareness, attitude and practice was 43.43%, 15.81% and 15.78%, respectively. The fitness of the proposed model among those constructs was confirmed by Root Mean Square Error of Approximation (RMSEA) <0.08, Normed chi-square (x 2/ df) < 5.0, Comparative Fit Index (CFI) ≥ 0.90, Tucker- Lewis Index (TLI) ≥ 0.9. Conclusion: The brucellosis prevention questionnaire has acceptable psychometric properties. Applying BPQ, veterinarians and educational planners can identify factors influencing preventive behavior of livestock breeders.