Percutaneous immune method is becoming an attractive alternative for DNA vaccine as a lot of antigen presenting cells are existed in the viable epidermis. However, due to the barrier function of stratum corneum, it would be hard for DNA vaccine to reach the viable epidermis of the skin. In order to deliver the DNA vaccine successfully cross the stratum corneum, pentagram silicon microneedle array was prepared in this study, and fluorescently labeled nanoparticle was taken as the model to observe the situation inside the skin processed by microneedle. Via microneedle nanoparticles could enter the skin through the micro-channel (diameter about 20–30 µm) and its amount is greatly larger than that enter though the hair follicle of intact skin. A new type of gene vector Pluronic P123-modified polyethyleneimine (P123-PEI) was synthesized by high molecular weight polyethylenimine and Pluronic P123 with the molar ratio of 1 : 1 to take the advantage of P123-PEI as low cytotoxicity and high transfection efficiency. Mice were immunized percutaneously with Hepatitis B DNA vaccine/P123-PEI nano-complexes by microneedle. The humoral and cellular immunity generated in percutaneously immunized mice through microneedle array by Hepatitis B DNA vaccine/P123-PEI nano-complex was significantly higher than that of DNA vaccine intramuscular administration.