ObjectiveTo prepare the ternary nano-gene polyplex by adding hyaluronic acid (HA) into DNA/polyamidoamine (PAMAM) binary nano-gene polyplex, and to probe into its gene transfection ability. MethodsDNA and HA were mixed with PAMAM dendrimer in different charge ratios to form ternary nano-gene polyplex, and the size and zeta potential of the polyplex were measured. MCF-7 and MDA-MB-231 cells were used for transfection efficiency test; B16 and MCF-7 cells were used for cell viability assay. Results and conclusionThe prepared ternary nano-gene polyplex has even size and stable structure. HA can enhance the gene transfection efficiency and reduce the cell toxicity compared with pure PAMAM, making it suitable for gene delivery and transfection.