The sustained-release formulation of protein has several unsolved problems, including the burst release and/or incomplete release of protein and the instability of protein during the preparation process. This study was conducted to find a way out in decreasing the burst release and incomplete release of protein as well as stabilising the protein. The method was to apply the solid-in-oil-in-water (S/O/W) encapsulation process. The different stabilising effects achieved by varying the proportion of zinc ion (Zn2+), protein and polyethylene glycol (PEG) during the preparation of Zn2+-bovine serum albumin (BSA) nanoparticles in PEG solution were investigated followed by the investigation of freeze-drying and subsequently encapsulating the nanoparticles mentioned above into polylactide-co-glycolide (PLGA) microspheres using the S-O-W method. The size-exclusion high-performance liquid chromatography results showed that the aggregation of protein released from the PLGA microspheres did not change significantly compared with that of the original BSA solution. The in vitro release results showed that burst release and incomplete release might be controlled through changing the proportion of zinc ion, protein and PEG in PLGA.