Reverse micellar extraction has a potential application in proteins purification. In the process of AOT/ isooctane reverse micellar extraction of proteins, a big problem is inactivation of interesting proteins when ionic surfactant AOT was used to form reverse micelles. In this study, it had been found that adding ethanol could stabilize the activity of protein, and it greatly improved extraction recovery of trypsin. When trypsin was extracted from crude material of pig pancreas by using AOT/isooctane reverse micellar system almost 90% and 100% of activity recovery of trypsin could be obtained in forward and backward extraction, respectively. The effects of ethanol concentration, organic salt types, AOT concentration, pH and temperature on the recovery of trypsin were investigated and the extraction process had been optimized. Finally, about 88% of the total yield was obtained, and the specific activity of trypsin purified was increased from 300 U/mg to over 1800 U/mg proteins with purification factor of 5 times more.