Type Ⅱ reovirus of grass carp represents the pandemic strain for grass carp hemorrahagic disease in China. VP56 is the outer most protein on the virion of type Ⅱ reovirus, and is suggested to play a key role during viral entry. To facilitate the study on biological functions of VP56, here, we investigated the prokaryotic and eukaryotic expression of VP56 gene of type Ⅱ reovirus. The VP56 ORF encoded by the S7 genomic fragment was amplified by RT-PCR technology from the cDNA of GCRV-JX02W infected CIK cells, and it was cloned into the pGEX-4T-3 and pFastBacHTA, respectively. After confirmation of the clones by sequencing analysis, the pGEX4T-3-VP56 was transformed into E.coli BL21(DE3) strain to express fusion protein GST-VP56. SDS-PAGE and Western Blot assays indicated that GST-VP56 was successfully expressed in E.coli BL21(DE3) strain with the molecular weight of about 83 ku. The recombinant protein mainly existed in the form of inclusion bodies. Polyclonal antibody was generated by immunization of mices with the purified recombinant VP56 proteins and the specificity of antibodies was determined by Western Blot. pFastBacHTA-VP56 was transformed into E.coli DH10Bac strain to get recombinant bacmid DNA that was analyzed by PCR. Then, the recombinant bacmid DNA was transfected into SF9 cells. Positive SF9 cells transfected with bacmid DNA stopped growth and lysed at 96 h post transfection.Western Blot results showed that the His-tag antibody could be specifically bound to His-VP56 fusion protein with the molecular weight of about 62 ku, which was soluble protein. In this paper, recombinant VP56 proteins were successfully expressed by both prokarotic and eukaryotic expression systems, and polyclonal antiserum against VP56 was generated, which laid a foundation for characterizing the function of VP56 protein during viral infection.