Objective To prepare a polyclonal antibody against S3 of Nelson Bay virus (NBV). Methods The E.coli BL21 (DE3) competent cells were transfected with the constructed Pris His MB-S3 recombinant plasmid. Protein expression was induced by IPTG. The target protein was purified by Ni-NTA column affinity chromatography to obtain a large amount of fusion recombinant protein, which was tested and used as the antigen for producing the S3 polyclonal antibody in rats. Results The relative molecular mass (Mr) of Pris His MB-S3 protein was around 39 000, in the form of inclusion bodies. NBV S3 polyclonal antibody was successfully produced from the immunized rats. The titer of the antibody was up to 1:64 000 determined by indirect ELISA. The indirect immunofluorescence assay verified that the S3 protein was successfully expressed in cells and distributed in a granular form in the cytoplasm. Conclusion The highly reactive and specific S3 protein polyclonal antibody is successfully prepared.