Objective To construct the prokaryotic expression plasmid of human mitoferrin 2 (SLC25A28), and to express and purify the protein for preparing its rabbit polyclonal antibody. Methods The prokaryotic expression plasmid pET28a(+)-SLC25A28-His was constructed and transferred into E. coli BL21 (DE3), and induced with Isopropyl-β-D-thiogalactopyranoside (IPTG). The SLC25A28 protein was extracted in form of inclusion bodies, and was further purified by His-NTA column after dissolved in 8 mol/L urea. The anti-SLC25A28 polyclonal antibody was prepared by immunizing rabbits, and its specificity was determined by Western blot analysis. Results pET28a(+)-SLC25A28-His was constructed and SLC25A28 protein was successfully expressed in E. coli BL21 (DE3) with the purity up to 90%. The Western blot results indicated that anti-SLC25A28 polyclonal antibody was capable to recognize specifically the SLC25A28 protein in testis. Conclusion The human SLC25A28 is successfully expressed in E. Coli, and the rabbit polyclonal antibody specific to SLC25A28 is prepared.