Nanotechnology represents a new impetus for biomedical research applications, especially using nanotechnology to formulate microspheres or nanospheres based delivery system for treatment of infectious diseases in animals. In this work, polylactic acid (PLA) microspheres with an average size of 156 nm were prepared by combining emulsion polymerization coupled with emulsion-solvent evaporation. Coating with polyethylenimine (PEI) polymers increased the surface charges of the resulting PLA/PEI microspheres, thus enabled plasmid DNA to adsorb tightly to the microspheres. As expected, the plasmid DNA was successfully transferred into the pig kidney-15 cells with high transfection efficiency. In addition, the protection rate of PLA/PEI microspheres loaded with DNA vaccine against foot-and-mouth disease in guinea pigs reached 87.5%, which was significantly higher than that of the pure DNA vaccine group. These results indicated that PLA/PEI microspheres were expected to be an effective delivery system for DNA vaccines.