Objective: To investigate microencapsulation technique, release of HBsAg-PLGA microspheres and degradation of the polymer in vitro, and the level of immune response after the single-dose HBsAg-PLGA microspheres subcutaneously injected (sc) to BALB/c mice. Methods: HBsAg-PLGA microspheres were prepared by double emulsion microencapsulation technique with orthogonal experiments. The pharmaceutical characteristics of size and surface morphology, antigen loading efficiency, release of HBsAg-PLGA microspheres and degradation of the polymer in vitro, and the level of immune response after single sc of PLGA microspheres in BALB/c mice were investigated. Results: The concentration of PVA was the significant factor affecting the particle size (P