Abstract: Objective To clone core sequence of hTERT promoter，and study transcriptional activity of hTERT promoter/SV40 enhancer in esophageal cancer cell. Methods hTERT promoter was amplified from human genomic DNA using polymerase chain reaction (PCR)；hTERT promoter was inserted into pGL3-Basic and pGL3-enhancer to construct luciferase gene expression driven by hTERT promoter(named as pGL3-hTERTp) or by hTERT promter/SV40 enhancer(named as pGL3-hTERTp-SV40en),respectively.The recombinants were transiently transfected into esophageal cancer cells of Eca-109，EC1 and human embryo lung fibroblast MRC-5, respectively，then expression level of luciferase gene in transfected cells was studied to evaluate transcriptional activities of hTERT promoter and hTERT promoter/SV40 enhancer in esophageal cancer cells. Results A 213 bp core-sequence of hTETR promoter was cloned successfully，and DNA sequencing showed its sequence the same as that registered in GenBank；recombinants of pGL3-hTERTp and pGL3-hTERTp-SV40en were successfully constructed.hTERT promoter had transcriptional activity in esophageal cancer cells while no transcriptional activity in MRC-5 cells；The transcriptional level of hTERT promoter/SV40 enhancer in esophageal cancer cells was significantly higher than hTERT promoter alone. Conclusion hTERT promoter has selective transcriptional activity in esophageal cancer cells；SV40 enhancer could significantly elevate transcriptional level of hTERT promoter，and it could play an important role in targeting gene therapy for tumor.