Abstract: VP39 is a protein encoded by S9 gene of type Ⅲ grass carp reovirus (GCRV-Ⅲ). In order to study the biological function of VP39 protein in the process of GCRV infection of grass carp cells, the sequence of VP39 gene was cloned and the prokaryotic expression vector PET32A-VP39 was constructed. The fusion protein VP39-HIS was obtained by using prokaryotic expression method. Mouse anti-VP39 polyclonal antibody was prepared by immunizing mice with VP39 solution protein, and the antibody was evaluated by Western Blot. The polyclonal antibody was used to investigate the expression dynamics of VP39 protein in GCRV infected cells. The results of SDS-PAGE showed that the fusion protein of vp39-his was well soluble in PBS and the protein size was about 39 kDa. Western Blot analysis showed that the prepared VP39 polyclonal antibody could recognize both the prokaryotic expression of VP39-HIS fusion protein and the expression of VP39 protein after GCRV infection with CIK cells at the dilution ratio of 1﹕10000, showing good titer and specificity. In the process of virus infection, the expression of VP39 was low in the early stage and high in the middle and late stage. Two peptides were screened by phage display technology for specific binding to VP39 protein, and further bioinformatics analysis also found that 7 genes in grass carp genome had homology with the peptide, indicating that these genes may interact with VP39. In this study, mouse anti-VP39 polyclonal antibody was prepared, which provided a new immunological method for GCRV-Ⅲ detection. The screening of binding peptides also laid a foundation for the study of the biological function of VP39 in the process of GCRV infection.