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Background: Considering the prevalence of brucellosis in Iran, it is necessary to choose a specific and sensitive laboratory method to diagnose it in a rapid and timely manner. The aim of this study was to assess the accuracy of indirect enzyme-linked immunosorbent assay (ELISA) for detecting brucellosis in humans in order to have an appropriate alternative to conventional tests such as Wright, 2ME, and commercial ELISA kits. Materials and Methods: In this study, the recombinant protein produced from the gene (omp28) bp26 Brucella melitensis was used as an antigen for coating microplate wells. A total of 124 serum samples of normal healthy individuals (n=62) and patients with acute brucellosis (n=62) approved by STA and 2 ME tests were entered into the study. The data were analyzed in SPSS (ver.18). Results: The mean age was 39.8±13.5 years in the patient group and 36.1±12.7 years in the healthy group. Furthermore, 66.1% of the patients were male and 62.9% lived in rural regions, while these figures were respectively 71% and 45.2% in the healthy group. The sensitivity of 92% and specificity of 87% and a positive predictive value of 88% and a negative predictive value of 92% and an accuracy of 90% were determined for ELISA kit used in this study. Conclusion: The ELISA diagnostic kit reacted to most of the positive human sera. However, this kit needs to be further evaluated with a larger sample size of clinical specimens from different regions and with various clinical forms of human brucellosis.