Brucellosis is a zoonotic bacterial infection caused by Brucella spp. and is more prevalent in the rural areas of Pakistan. Serological tests’ including Rose Bengal Plate Test (RBPT) is gold standard method for the diagnosis of Brucellosis. In this study the RBPT antigen was prepared, and its efficiency was compared with the local and imported RBPT antigens.  A total of 288 serum samples from different animals were collected. In-house RBPT antigen was prepared, and its efficiency was compared with the local and imported RBPT antigens using serum agglutination test (SAT) and competitive enzyme linked immune sorbent assay (cELISA).  The levels of concordance (288 sera) of in-house with local and imported RBPT antigens were 80.2% and 80.5% respectively. A total 174 sera were tested by cELISA which showed 82.7% concordance between cELISA and in-house RBPT antigen. The concordance level of cELISA with local and imported antigen was 77% and 72.9% respectively. The relative sensitivities by local and imported RBPT antigens were 66.1% and 62.3% and relative specificities were 81.3% and 78.1% respectively. The relative sensitivity (84.9%) and specificity (82.1%) of in-house RBPT antigen was found higher as compared to other available antigens. The local, imported and in-house RBPT showed 74.2 (95% CI, 65.8 – 82.6), 70.2 (95% CI, 61.4 – 78.9) and 83.9 (95% CI, 77.1 – 90.7) area under the receiver operating characteristic (AUROC) curve. The newly prepared RBPT antigen is more efficient in terms of sensitivity and specificity for brucellosis diagnosis.