Objective: To construct the U6/H1 dual promoter siRNA expression cassettes (SEC) targeting human telomerase hTERT gene, and to observe the interfering effect of their transcription products on hTERT gene activity in HeLa cells. Methods: By fusion PCR, three U6/H1 dual promoter SECs targeting the open reading frame of the hTERT gene and one SEC targeting the 3’ untranslated region of the hTERT gene were constructed. The SECs were transferred into human cervical cancer HeLa cells respectively after identification, and then the telomerase activity in the HeLa cells was assessed by telomeric repeat amplification protocol (TRAP). Results: The four SECs targeting human telomerase hTERT gene were all successfully constructed, and after their transfection into the HeLa cells, the rate of telomerase activity inhibition was 36.8%, 57.39%, 80.47%, and 70.31%, respectively. Conclusion: The successful construction of SECs targeting human telomerase hTERT gene may provide a novel effective approach for study of the gene interference of tumor telomerase.