Abstract: Extraction and separation of glucamylase by two-phase aqueous systems was investigated. The effects of the molecular weight (600, 800, 1000, 2000) and concentration of polyethylene glycol (PEG) , the concentrations of salt (MgSO4, (NH4) 2SO4, KH2PO4) and the four crude enzymes of glucamylase (Rhizopusoryzae, Rhizopus, Distillery yeast, Esterification yeast) on the purification factor were studied. The results showed that Rhizopusoryzae under the PEG1000's mass concentration of 15%, MgSO4 with the concentration of 22.86% got the purification factor were 3.35. Distillery yeast and Esterification yeast at the PEG1000's concentration with15%, the (NH4) 2SO4's concentration were 15.73% and 13.33%, respectively, got the purification fold were1.82, 1.50. Rhizopus at the molecular weight of PEG with 2000, concentration was 15%, the (NH4) 2SO4's concentration was 14.37%, The purification fold were 2.07. This method could keep biological activity and simplicity of operator, had good application prospects.