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Background and aim: Brucellosis is one of the world most common zoonotic diseases. The current study was aimed to prepare the Rose Bengal Plate Test (RBPT) antigen for the diagnosis of brucellosis and to determine its specificity and sensitivity. Material and methods: The Rose Bengal plate test antigen prepared from Brucella abortus (B. abortus) strain 99 was compared with two commercial Rose Bengal Plate Test antigens and its specificity and sensitivity are determined. Results: The were Rose Bengal plate test and I-ELISA result show that the in vitro antigen was superior to RBPT antigen University Diagnosis Laboratory (UDL) Lahore Pakistan, and RBPT antigen Veterinary Laboratory Agency (VLA) UK. Out of 196 samples analyzed by in vitro RBPT antigen, RBPT antigen (UDL), RBPT antigen (VLA), and an indirect enzyme-linked immunosorbent assay (I-ELISA) 56.63 %, 53.57%, 41.84%, 35.71% were found B. abortus positively. The sensitivity calculated for the in vitro RBPT antigen was 96.62, while RBPT antigen (UDL) and RBPT antigen (VLA) were 89.77, 63.91 correspondingly. However, the specificity of the in vitro RBPT antigen was lower (77.57%), than the commercial RBPT antigen (VLA) (79.79%). Conclusions: A very sensitive and low-cost in vitro RBPT antigen compared to commercial RBPT was magnificently developed in the current study. It was determined that the in vitro RBPT antigen could substitute the available commercial RBPT antigen, which is comparatively expensive and less sensitive in the detection of brucellosis. Therefore, it is concluded that the in vitro RBPT antigen could be used for epidemiological surveillance of brucellosis.