Optimal preparation conditions of DNA vaccine against swine influenza encapsulated in Poly (D, L)-lacticco-glycolic acid (PLGA) microspheres were determined. The microspheres were prepared by an emulsion-evaporation method using PLGA as the biodegradable matrix forming polymer. Using the optimal preparation conditions, PLGA microspheres containing the DNA vaccine were produced with good morphology as evident from scanning electron micrographs, high encapsulation rate and high stability. The transfection test indicated that the vaccine could be expressed as an antigen in cells and maintained good bioactivity. Moreover, these results demonstrated that the PLGA microspheres containing DNA vaccine can be used to achieve prolonged release of plasmid DNA. These results have laid a foundation for further development before ultimate industrial application.