BSA-loaded mcirospheres were prepared by a modified phase separation method, in which petroleum ether (PE) containing a certain amount of Span 80 rather than poly (dimethylsiloxane) (PDMS) was adopted as coacervating agent. Process parameters such as Span 80 concentration, the volume and addition rate of coacervating agent, polymer concentration, agitation rate during the phase separation process and PE type were evaluated to optimize the protein encapsulation. It was found microspheres with high yield (>80.0%) and entrapment efficiency (>90%) could be obtained using PE containing 5.0% Span 80 as the coacervating agent. Microspheres with small particle size (<10 microm) could be produced successfully with appropriate process parameters. In vitro release study suggested that burst release was significantly influenced by Span 80 concentration, polymer concentration and PE type and the burst release could be reduced to <20% with optimized formulation. A biphasic release behavior in vitro test was observed for the microspheres prepared by this method. GC analysis demonstrated that residual solvent of DCM and petroleum ether was decreased dramatically in comparison with PDMS used as a conventional coacervating agent.