Objective:To construct the prokaryotic plasmid expressing the S100A16 and prepare the rabbit polyclonal antibody against S100A16. Methods:S100A16 gene was amplified by RT-PCR and cloned into the expression vector pET-28a,then the recombinant plasmid was transformed into E. coli BL21 (DE3) and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and identified by SDS-PAGE. The rabbit was immunized with fusion protein to produce polyclonal antibody,and the sensitivity and specificity of the antibody was evaluated by enzymelinked immunosorbent assay (ELISA) and Western blot. Results:The recombinant expression plasmid has been successfully constructed,which was confirmed by the restriction enzyme digestion and DNA sequencing analysis. The recombinant protein could be expressed effectively,and the antibody was specific and effective. Conclusion:The S100A16 prokaryotic expression plasmid was constructed,and polyclonal antibody directed against this protein has been successfully prepared,which will provide a useful tool for the further research of S100A16.