Abstract: In order to solve the problem of low adenovirus-transfected efficiency on cancer cells of tumor gene therapy, efficient and low-toxicity transfection reagents have been considered as a promising method. In this research, n-Dodecyl-β-D-Maltoside (DDM), DC-Chol and SDS were affected synergistically with eGFP-Ad to transfect HepG2, EC109, L-02 and Het-1A cells. The drug effects were evaluated by detection and comparison of the green fluorescence expression after 48 hours of adenovirus transfection. The data showed that the transfection efficiency of DDM, DC-Chol, and SDS was significantly improved in HepG2 and EC109 cancer cells when comparing to L-02 and Het-1A human normal cells. The incremental transfect efficiencies of DDM (3 μg/mL), DC-Chol (1 000 μg/mL) and SDS(0.5 μg/mL) on tumor cells HepG2 and EC109, at about 85% and 78%, 80% and 76%, 45% and 41%, were 16 times and 14.6 times, 15 times and 14.2 times, 8 times and 7.2 times as great as the non-treated groups. The results indicated that DDM significantly improved the transfection efficiency of adenovirus on tumor cells, which can provide the technical reference for adenovirus-mediated gene therapy.