Objective:To construct the fusion gene of human hTERT and human heat shock protein 70(HSP70), and expressed it in E.coli. Methods:The hTERT and HSP70 genes were amplified by PCR. The hTERT gene was digested by NcoⅠand EcoRⅠ enzyme and inserted into the plasmid pET32a to construct pET32a-hTERT plasmid. Then the HSP70 gene was cloned into the expression vector pET32a-hTERT between EcoRⅠ and HindⅢ site to construct the prokaryotic expression plasmid pET32a-hTERT-HSP70. The E.coli BL21 contained the plasmid was induced by IPTG. Results:The HSP70 gene and hTERT gene segments were amplified from their templates; the DNA sequencing result showed that the prokaryotic fusion expression plasmid pET32a-hTERT-HSP70 was successfully constructed. The E.coli BL21 contained the plasmid could express a Mr 110 kD protein after induced by IPTG. Conclusion:The successful expression of fusion protein of human hTERT and heat shock protein 70 enables the further research of the HSP70 protein as a adjuvant-free carrier.