High efficiency and serum resistant capacity are important for gene carrier in vivo usage. In this study, transfection efficiency and cell toxicity of polyethylenimine (PEI) (branched, Mw = 25K) was remarkably improved, when mixed with polyanion (polyethylene glycol-polyglutamic acid (PEG-PLG) or polyglutamic acid (PLG)). Different composite orders of PEI, polyanion, and gene, for example, PEI is first complexed with DNA, and then with polyanion, or PEI is first complexed with polyanion, and then with DNA, were studied. Results showed that only the polyanion/PEI complexes exhibited additional properties, such as decreased pH, resulting in increased particle size, as well as enhanced serum resistance capability and improved tumor accumulation. The prepared gene carrier showed excellent antitumor effect, with no damage on major organs, which is suitable for in vivo gene antitumor therapy.