Objective:To construct a prokaryotic expression vector pCold－Ⅱ－Rap and to optimize the expression conditions of ranpirnase (Rap) protein and to determine the protein activity. Methods:Recombinant pCold－Ⅱ－Rap vector was constructed,and positive clone was selected and transformed into E.coli.BL21. The recombinant protein expression was induced by IPTG. The Rap recombinant protein was purified,and detected by SDS－PAGE and Western blot. The RNase activity of Rap was analyzed by MTT. Results:The results showed that the purified ranpirnase was obtained by optimizing conditions of expression and purification, and degradation of tRNA could be detected by ranpirnase in vitro. The fusion protein can inhibit the proliferation of breast cancer cell MDA－MB－231,the inhibition ratios were 93.49% at the 4th day. Conclusion:The recombinant ranpirnase would be applied as a potential therapeutic drug,which could conjugated with a tumor specific antibody or antibody fragment.