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The preparation of capillary electrochromatography column for chiral separation with β-cyclodextrin derivatized Pancreatin was investigated, which had three steps, synthesis ofβ-cyclodextrin derivated pancreatin, the modification of the capillary internal surface and the covalent binding of β-cyclodextrin derivated pancreatin on the capillary internal wall. β-cyclodextrin derivated pancreatin was synthesized using β-cyclodextrin and protein in the presence of ethylene glycol diglycidyl ether (EGDE) in boric acid buffer at pH = 8.7. Amino group was coated on the internal surface of the silica capillary by sol-gel technology using triethoxylmethylsiloxane and (3- aminopropyl ) trimethoxysiloxane . Covalent binding of β- cyclodextrin derivated pancreatin was performed by glutaraldehyde. Chlorpheniramine, phenylalanine and troopine were separated baseline by β-cyclodextrin derivated pancreatin in capillary electrochromatography.