The aim of this study is to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres containing a staphylokinase variant K35R (DGR) with purpose of preserving the protein stability during both encapsulation and drug release. DGR-loaded microspheres are fabricated using a double-emulsion solvent extraction technique. Prior to encapsulation, the effect of ultrasonication emulsification of DGR solutions with methylene chloride on protein recovery was investigated. Moderate ultrasonic treatment of aqueous DGR/dichloromethane mixtures caused approximately 84% DGR aggregation. Polyvinyl alcohol (PVA) added into aqueous DGR solutions significantly improved DGR recovery to >90%. The effects of co-encapsulated PVA and NaCl in the external aqueous phase on the characteristics of the microspheres were investigated. When 2% PVA was co-encapsulated and 2.5% NaCl was added to the external water phase, DGR encapsulation efficiency was significantly increased from 7.1% to 78.1% and DGR was distributed uniformly throughout the microspheres. In vitro release test showed that DGR was released from PLGA microspheres in a sustained manner over 15 days. A large amount of released DGR was inactive in the absence of co-encapsulated PVA. On the contrary, when 2% PVA was co-encapsulated, the released DGR was almost completely intact within 9 days. In conclusion, PLGA microspheres can be an effective carrier for DGR and form a promising depot system.