To investigate the RNA-dependent RNA polymerase (RdRp) gene of Chinese sacbrood virus (CSBV), a serious threat to the health of Apis cerana cerana, its expression in prokaryotic cells and the preparation of corresponding polyclonal antibodies, the full-length sequence of RdRp gene was amplified by PCR, cloned into the pET-28a prokaryotic expression vector, and then introduced into Escherichia coli Rosetta (DE3) cells. The target protein was induced to express in large quantities by IPTG-β-D-thiogalactoside, and polyclonal antibodies were prepared by immunizing mice (Mus musculus). The specificity of the polyclonal antibody was analyzed by Western blot. The results showed that the recombinant expression plasmid pET28a-RdRp was successfully constructed, and large amounts of RdRp recombinant protein were expressed in E. coli Rosetta (DE3) cells. The Western blot analysis results showed that the polyclonal antibody prepared could specifically recognize RdRp in the larvae of Apis cerana cerana infected with CSBV. The above research results lay a foundation for further studying the precise location of RdRp and revealing the specific process of CSBV invading host cells.