Three-phase partitioning used salt and an organic solvent to precipitate enzymes or proteins from complex mixtures. Three-phase partitioning was applied to prepare β-glucosidase from bitter almond crude extract. In order to enhance the purification fold degree and activity recovery of β-glucosidase, the study discussed the influence of organic solvents, organic solvent concentration, ammonium sulfate concentration, pH values and temperature on the parameters of β-glucosidase partitioning process. The results were shown below: t-butanol was the best organic solvent, the ratio of crude extract and t-butanol was 1.0:1.5 (v/v), ammonium sulfate saturation was 50% (w/v), the system of pH value was 5.0 and the optimum temperature was 25℃. Under the optimized conditions, the β-glucosidase was purified to 5.97-fold with 85.7% activity recovery. The SDS-PAGE of the enzyme analyzed that comparatively purification and protein molecular weight of the enzyme was found to be about 60.0 kDa. This study showed that three-phase partitioning is a simple, quick and economical method, which was applied to β-glucosidase separation and purification.