Bovine brucellosis is endemic in many parts of the world including India. The disease diagnosis and surveillance are usually carried out by serological tests, which however have drawbacks. This study was undertaken to evaluate the potential of real-time PCR (RT-PCR) targeting bcsp31 gene for surveillance of bovine brucellosis. A total of 461 samples, which included 408 stored serum and 53 prospective blood samples, were used. It was found that 33 (7.15 %) samples were positive by RT-PCR, whereas 149 (32.32 %) and 132 (28.63 %) were positive by Rose Bengal plate test (RBPT) or standard agglutination test (STAT), respectively. The results of this study suggest that RT-PCR targeting bcsp31 gene carried out on DNA extracted from serum or blood may not be a suitable method for surveillance of brucellosis in bovines.