The goal of the present paper was to optimize the encapsulation of beta-lactoglobulin (BLG) within poly(lactide-co-glycolide) (PLGA) microparticles prepared by the multiple emulsion solvent evaporation method. The role of the pH of the external phase and the introduction of the surfactant Tween 20, in the modulation of the entrapment and release of BLG from microparticles, was studied. Reducing the solubility of BLG by decreasing the pH of the external phase to a value close to the pI of BLG resulted in a better encapsulation with, however, a larger burst release effect. By contrast, Tween 20 was shown to increase the encapsulation efficiency of BLG and reduce considerably the burst release effect. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface. In addition, Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communicating with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease in the burst release. These results constitute a step ahead in the improvement of an existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method.