Brucellosis is an emerging zoonotic disease caused by members of the genus Brucella and remains a serious cause of human illness fromlivestock. Presently used sero-diagnostic tests depend upon the smooth lipopolysaccharide (sLPS), which cross react with other GramÂnegative bacteria resulting in lowspecificity. To overcome this, several recombinant outer membrane proteins (OMP) have been tried as diagnostic antigen (s) in ELISA. In the present study, the ORF (753 bp product) of BP 26 protein was amplified fromBrucella suis strain 1330, cloned in pET32a vector and expressed in BL21 E.coli hostcell. The expressed protein was purified by Ni-NTAcolumn and characterized by SDS-PAGE andWestern blot analysis. The purified recombinant protein (rbp26) antigen was tested in indirect ELISA (iELISA) and specificity was checked with E. coli (O157 H7), 17 salmonella and five Yersinia entericolitica reference sera. Further, rbp26 based standardized ELISA was evaluated with serum samples (n=626) collected fromrisk group individuals (veterinarians) using two conjugates IgM and IgG for diagnosis of brucellosis. Comparative evaluation of the developed assay with RBPT and sLPS based ELISA was carried out. In RBPT, 60 (9.5%) and in sLPS antigen based iELISA-IgM36 (5.75%) and iELISA-IgG 122 (19.48%) were positive, respectively.Whereas in rbp26 based ELISA 18 (2.87%) and 66 (10.54%) were positive in IgM and IgG ELISA, respectively.