Objective：To express the granulysin protein in E. coli and to detect its bioactivity after purification. Methods： Using cultured human peripheral blood mononuclear cells as the template, we selectively amplified the fragment coding granulysin by RT-PCR. The fragment was then inserted into the prokaryotic expression vector pET28a （＋） for transforming E. coli BL21（DE3） plysS. Fusion protein expression was induced by isopropyl-Beta-D-thiogalactopyranoside （IPTG）. After renaturation, the protein was purified by affinity chromatography and its bioactivity was examined by colony-forming unit. Results： We successfully inserted granulysin cDNA fragment into vector pET28a （＋） and constructed the expression plasmid pET28a （＋）GNLY. The fusion protein, with a molecular weight of 9 000, was obtained through IPTG induction. After renaturation and purification, the recombinant protein was proven to be bioactive by CFU and its cytotoxicity was in a dose-dependent manner. Conclusion： The fusion protein obtained in the present study is bioactive and can be used for further study on its function,mechanism of action and clinical application.