In recent years, the frequent outbreaks of viral hemorrhagic diseases have seriously affected the development of grass carp (Ctenopharyngodon idella) aquaculture, but its pathogenesis is still unclear. The viral hemorrhagic disease of C. idella is caused by grass carp reovirus (GCRV). Type II GCRV, as the main epidemic strain at present, is highly pathogenic to C. idella and has a high lethality rate. The VP35 protein encoded by the fragment S11 of type Ⅱ GCRV is the outer capsid protein. VP35 protein may play an important role in binding to receptors to promote virus entry into host cells, and may also promote virus replication by interacting with intracellular proteins. In order to explore the role of VP35 protein in virus replication, the S11 gene was amplified and inserted into the vector pCMV-3×flag to construct the S11 plasmid, which was transfected into C. idella kidney (CIK) cells, and then infected with type Ⅱ GCRV. After infection, the supernatant and cells were collected. First, real-time fluorescent quantitative PCR was used to detect S6 mRNA in cell samples. The results showed that overexpression of VP35 promoted the replication of type Ⅱ GCRV at the level of viral mRNA by about two-fold. Then the S6 gene was amplified and inserted into the vector pET-32a (+) to construct a recombinant plasmid, and the recombinant protein His-VP4 were purified and polyclonal antibodies was prepared to determine the effects of VP35 on type Ⅱ GCRV at viral protein level. The results showed that overexpression of VP35 promoted the replication of type Ⅱ GCRV at the level of viral protein. Finally, the pET-32a-S6 plasmid was used as a positive standard plasmid to establish an absolute quantitative detection method for the gene copy number of type Ⅱ GCRV. The pET32a-S6 plasmid was diluted 10-fold, and then used as a template, qPCR was used to detect the expression of the S6 gene, and a standard curve was drawn. The quantitative method was used to determine the effects of VP35 on type Ⅱ GCRV particles in supernatants. The results showed that overexpression of VP35 increased the copy number of type Ⅱ GCRV. This study proved from three dimensions that overexpression of VP35 protein promoted type Ⅱ GCRV replication, which is helpful for in-depth analysis of the function of VP35 protein, and laid a foundation for the subsequent exploration of the mechanism of VP35 protein in promoting virus proliferation, provided a protein-level detection method for further study of type II GCRV virus, and a new technical means for rapid and accurate quantification of virus titer.