Grass carp reovirus (GCRV) has been considered as the most pathogenic agent and poses a significant threat to grass carp culture. A virulent reovirus strain, HZ08, was isolated from diseased grass carp in Zhejiang province, and has been proven to be epidemic strain in China. To development serological methods for detecting prevalent GCRV, the gene encoded for major outer capsid VP4 was selected clone to plasmid pET32a( ) and the purified recombinant VP4 protein was injected into BALB/c mice through subcutaneous route. The spleen cells from immuned BALB/c mice were fused with SP2/0 myeloma cells, and three hybridoma cell lines, designated as 2C2, 2F3 and 5E5 respectively, were screened out to be able to secret monoclonal antibody (McAb) against VP4 protein using indirect ELISA. All the McAb were IgG1 subtype with κlight chain and could not react with GSRV, ISKNV, IHNV except GCRV-HZ08. The hybridoma cell line 2C2 was selected for McAb preparation, and the titers in cell culture medium of the ascetic fluids were up to 1:720,000. The results of western blot and IFA showed that the McAb could recognize the authenticVP4 protein of GCRV HZ08 particles. In present study, the McAb against VP4 of GCRV HZ08 was successfully prepared, which laid a foundation of developing a rapid determination method for GCRV and further study of structures and functions of VP4 protein.