The use of chitosan for gene delivery is limited due to the low transfection efficiency and difficulty in transfecting into a variety of cell types, especially the hepatoma cells. In order to solve this problem, lactobionic acid (LA) bearing galactose group was coupled with water-soluble chitosan (WSC) for liver specificity and poly(ethylenimine) (PEI) was combined to galactosylated chitosan (GC)/DNA complexes to enhance the transfection efficiency. For initial study, the effect of PEI on the transfection efficiency of WSC/DNA complex was studied in HeLa, A549 and 293 T cells, and bafilomycin A1 was used to ascertain the mechanism of synergistic effect. Transfection efficiency, cytotoxicity, and physicochemical properties of GC/DNA complex combined with PEI were investigated to determine the potential for the hepatocyte-targeting. The combination of PEI with WSC/DNA and GC/DNA complex dramatically increased the luciferase expression 10- to 1000-fold in various cell lines, and the synergistic effect was proved to be induced by proton sponge effect of PEI. The transfection of GC/DNA complex in HepG2 was much higher than that of WSC/DNA even after combination with PEI, and was highly inhibited in the presence of galactose. Cytotoxicity of PEI was much decreased by combination with GC/DNA complex. And PEI was proved to be coated on the surface of GC/DNA complex through the ionic interaction.