Brucellosis is a major bacterial zoonosis of global importance. The conventional serological tests viz. RBPT, SAT and CFT have certain inherent problems which are circumvented with the development of ELISA. But till date species specific conjugate required for ELISA in buffaloes is not available. In the present study, Fluorescence Polarization Assay was standardized using indigenously prepared antigen for the diagnosis of brucellosis in buffaloes. The OPS was extracted from B. abortus S99 and conjugated with FITC to obtain FPA antigen. The fluorescence intensity of the FPA antigen was adjusted between 2,50,000 and 3,00,000. The cutoff for FPA was determined to be 116.8mP units using ROC analysis program of MedCalc Software. The sensitivity, specificity and P.I. of FPA were 92.95%, 98.17% and 191.12, respectively and were comparable to those observed with anti-bovine ELISA and Protein-G ELISA. The sensitivity and P.I. values of FPA were much higher than SAT and RBPT, however specificity values of FPA, SAT and RPBT were comparable. The Kappa test revealed perfect agreement between FPA, anti-bovine ELISA and Protein-G ELISA and only a substantial agreement between FPA and RBPT.