DNA vaccination has great potential to treat or prevent avian influenza pandemics, but the technique may be limited by low immunogenicity and gene delivery in clinical testing. Here, to improve the immune efficacy of DNA vaccines against avian influenza, we prepared and tested the immunogenicity of 4 recombinant DNA vaccines containing 2 or 3 AIV antigens. The results revealed that chickens and mice immunized with plasmid DNA containing 3 antigens (HA gene from H9N2, and NA and HA genes from H5N1) exhibited a robust immune response than chickens and mice immunized with plasmid DNAs containing 2 antigenic genes. Subsequently, this study used pβH9N1SH5 as a model antigen to study the effect of dendritic polylysine (DGL) nanoparticles as a gene delivery system and adjuvant on antigen-specific immunity in mice models. At a ratio of 1:3 DGL/pβH9N1SH5 (w/w), the pβH9N1SH5/DGL NPs showed excellent physical and chemical properties, induced higher levels of HI antibodies, and larger CD3+/CD4+ T lymphocyte and CD3+/CD8+ T lymphocyte population, as well as the production of cytokines, namely, interferon (IFN)-γ, interleukin (IL)-2 compared with the naked pβH9N1SH5. Therefore, multiantigen gene expression methods using DGL as a delivery system may have broad application prospects in gene therapy.