Insulin-loaded poly(lactide-co-glycolide) nanoparticles (INS-PLGA-NPs) were prepared by a double emulsion method (w/o/w), using ethyl acetate as organic solvent and poloxamer188 as emulsifier. Experimental parameters such as the emulsifier and PLGA concentrations, the pH and concentration of the insulin solution, the solvent evaporation method and PVA in the internal phase were investigated for the encapsulation efficiency. The results indicated that higher emulsifier concentration, relatively less amount of PLGA and lower insulin concentration would increase the entrapment of insulin. Furthermore, pH of insulin solution approaching to pI (5.3), adding some PVA to the internal phase and a shorter evaporation time helped to enhance the incorporation efficiency of insulin. Optimized preparation parameters led to nanoparticles with well-defined characteristics such as an average size around 149.6 nm, a polydispersity lower than 0.1 and high encapsulation efficiency up to 42.8%.