Macroporous chitosan layer coated on silica gel (CTS-SiO2) was prepared by phase inversion and polyethylene glycol (PEG) molecular imprinting methods. Effects of chitosan and PEG content on the formation of porous surface were investigated by scanning electron microscopy (SEM). Trypsin could be covalently coupled as an affinity ligand on the macroporous chitosan layer activated with epoxy groups. The activity of the immobilized enzymes in the optimum conditions was 594 +/- 8 U per gram CTS-SiO2 bead, which account for 47.5 +/- 0.6% of original trypsin activity in the incubation solution. In comparison with the free enzyme, the immobilized enzyme showed high stability at room temperature with less than 10% activity loss in 7 days storage. Moreover, non-specific adsorption was not observed on this kind of affinity chromatographic matrix. The immobilized trypsin bead was packed into column to separate the trypsin inhibitors (TIs) from egg white. The recovery rate of the trypsin inhibitor activity was 54.5 +/- 1.2% and purification folds could reach 9.1 after one step of chromatographic process. (c) 2005 Elsevier B.V. All rights reserved.