Objective: To construct the artificial miRNA expression cassettes targeting hTERT gene and validate their inhibitory effect on telomerase activity in HepG2 cells. Methods: Three artificial miRNA expression cassettes targeting different sites of hTERT gene were designed and constructed by overlap extension PCR method. After identification, the cassettes were transfected alone or co-transfected into HepG2 cells, and then the telomerase activities in HepG2 cells were detected by TRAP-silver staining and TRAP-duplex scorpion probe fluorescence quantitative PCR. Results: The three artificial miRNA expression cassettes targeting hTERT gene were all successfully constructed, and after they were transfected alone or co-transfected into the HepG2 cells, the telomerase activities in HepG2 cells were inhibited to different degrees, and the inhibitory effect of co-transfection with two miRNA expression cassettes was significantly stronger than those of any lone transfection. All the differences reached statistical significance (all P<0.05). Conclusion: The artificial miRNA expression cassettes targeting hTERT gene can effectively inhibit telomerase activity in HepG2 cells, and combined inhibition is an effective experimental program.