To express the Listeria monocytogenes hly gene in Escherichia coli and study its primary biological characteristics, hly gene without signal peptide sequence was amplified from Listeria monocytogenes serotype 4b by PCR and inserted into T-easy vector. Sequencing showed this hly gene was 1518 bp and nucleotide homology was more than 99.8% compared with three Listeria monocytogenes hly genes in GenBank. The cloned hly gene was then inserted into prokaryotic expression vector pGEX-6P-1 and transformed into E. coli BL21. The predicted fusion protein was detected by SDS-PAGE after IPTG inducion, which had molecular weight approximately 82 kD. Hemolytic experiment demonstrated the expressed fusion protein can lyse human red cell and its hemolytic titer attained 2.26 x 10(4) HU/mg. Conclusively, the Listeria monocytogenes hly gene was successfully cloned and expressed in E. coli. The successful expression of LLO in E. coli BL21 constituted a solid foundation for further researches such as pathogenesis and immune mechanism, MAb and vaccine development.