Background and Objectives: Brucellosis is an infectious zoonotic disease that continues to be of public health and economic concerns in many parts of the world. Since Iran is an endemic area for brucellosis, this study was conducted to look into the sero-prevalence of brucellosis in blood donors in Khuzestan province, South West of Iran.Materials and Methods: In the cross-sectional study, 3000 sera were obtained from blood donors having referred to transfusion centers of Khuzestan cities in 2011. Sera were screened for serological evidence of brucellosis using the Rose Bengal Plate Test (RBST), Standard Tube Agglutination Test (STAT), and the 2-Mercapto ethanol (2ME) test. A titer of³1.80 for STAT and a titer of³1.40 for 2ME were taken as criteria of brucellosis. Data were analyzed by SPSS 18 software and Chi test.Results: Seropositivity rates of brucellosis in blood donors were 1.7%, 1.46%, and 0.56% by RBPT, STAT, and 2ME, respectively. There was no statistical correlation between brucellosis and sex or age groups (p=0.26 and p = 0.56, respectively). The highest rate of brucellosis sero-prevalence was found in Izeh (1.7%) and the lowest in Ahvaz (0.2%). The area of living and season were significantly associated with brucellosis (p = 0.032 and p = 0.019, respectively).Conclusions: Based on the results, the Seroprevalence of brucellosis in blood donors at Khuzestan province is greater than expected.