Trypsin inhibitor was converted to hydrophobic states by covalently combining cholesteryl groups using an acylation reaction, and was immobilized in reverse micelles composed of a nonionic surfactant. Using this reverse micellar phase containing trypsin inhibitor as an affinity ligand, trypsin was selectively separated with high recoveries from a mixture of several kinds of contaminating proteins by forward and backward extraction. No loss of activity of the recovered trypsin was observed through these operations.