A pair of primers were designed according to the nsp5 gene of human group B rotavirus strain WH-1, and the nsp5 gene was amplified by PCR and subcloned into the expression vector pGEX-KG. After induced with IPTG, the NSP5 was expressed as a GST fusion protein in the soluble form. Then the protein was purified and immunized mice to obtain specific antiserum. At last, polyclonal antibody was analysed by Western Blot and the result indicated the specification and high titer of the antibody. This work provides important information of the immunogenicity and possible function exploration of NSP5, which is helpful for futher study on virus replication and pathogenic mechanism.