Gold particles have been used as a carrier for transdermal gene delivery, which may cause adverse side effects when accumulated. In this study, biodegradable nanoparticles, composed of chitosan (CS) and poly-gamma-glutamic acid (gamma-PGA), were prepared by an ionic-gelation method for transdermal DNA delivery (CS/gamma-PGA/DNA) using a low-pressure gene gun. The conventional CS/DNA without the incorporation of gamma-PGA was used as a control. Small-angle X-ray scattering (SAXS) was used to examine the internal structures of test nanoparticles, while identification of their constituents was conducted by Fourier transformed infrared (FT-IR) spectroscopy. The CS/gamma-PGA/DNA were spherical in shape with a relatively homogeneous size distribution. In contrast, CS/DNA had a heterogeneous size distribution with a donut, rod or pretzel shape. Both test nanoparticles were able to effectively retain the encapsulated DNA and protect it from nuclease degradation. As compared with CS/DNA, CS/gamma-PGA/DNA improved their penetration depth into the mouse skin and enhanced gene expression. These observations may be attributed to the fact that CS/gamma-PGA/DNA were more compact in their internal structures and had a greater density than their CS/DNA counterparts, thus having a larger momentum to penetrate into the skin barrier. The results revealed that CS/gamma-PGA/DNA may substitute gold particles as a DNA carrier for transdermal gene delivery.