Abstract: Trypsin is an important proteolytic enzyme，already used in large-scale processes by the detergent and dairy industries. Separation of trypsin directly from porcine pancreatic homogenate in aqueous two-phase systems (ATPS) of polyethylene glycol/ammonium sulfate was studied．The effects of polyethylene glycol 400 (PEG400) concentration，(NH4)2SO4 concentration，NaCl concentration，and pH value on the extraction of porcine pancreatic trypsin were investigated in terms of phase volume，partition coefficients of both enzyme activity and protein，and recovery．Orthogonal experiment was used to further analyze and optimize the separation of trypsin. The results indicated that the control of the concentration of (NH4)2SO4 and PEG400 were the most important for partitioning of trypsin．The NaCl addition had little effect on trypsin partition. Maximum partition coefficient of enzyme activity was 8.48 under the optimum separation conditions of 24% PEG400，20% (NH4)2SO4，and pH 4.2. The separation was successfully scaled up to 100 g system with the trypsin activity of 1，780 U/ mL at 20℃.