A novel strategy for patterning several kinds of proteins in a specific position of a microchannel is presented. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are covalently immobilized on a polystyrene microchannel surface through a photoreactive crosslinker with an azide group at one end and a succinimide group at the other by UV irradiation. The activities of HRP and ALP are evaluated using Amplex Red and Fluorescein diphosphate, respectively. The fluorescence has been observed only at the enzyme-immobilized position.