In this study, a novel nonviral gene delivery system, which could enhance the inhibition effect of antisense oligonucleotides (ASODN) against the tumor cells, was developed. The polycation liposomes (PCLs) were prepared using the film hydration method with dioleoylphosphatidylethanolamine (DOPE) and amphipathic compound polyethylenimine-cholesterol (PEI 800-Chol), synthesized by low-molecular-weight polyethylenimine (PEI, MW 800) covalent conjugation with cholesterol. The formation of PEI 800-Chol was confirmed by IR and critical micelle concentration detection. The transfection efficiency of PCLs mediating Green Fluorescence Protein plasmid (pEGFP) in HeLa cells was evaluated and the highest gene expression was obtained by PCLs containing DOPE, which was 1.6-fold of that induced by commercial Lipofectamine 2000, and the gene expression efficiency was influenced in the present of serum. Subsequently, human telomerase reverse transcriptase gene antisense oligonucleotides (hTERT-ASODN) were used as therapeutic gene, and the results showed that PCLs, which demonstrated very low cytotoxicity itself, could significantly enhance the inhibition efficiency of hTERT-ASODN in the growth of tumor cells. These results suggested that the PCLs could be widely applied for ASODN delivery.