A microfluidic chip with in-situ prepared chitosan membrane immobilized with Glucose Oxidase was developed as glucose oxidase reactor to detect human serum. The chitosan was prepared in-situ in the micro-channel as the immobilization material.Then,GOD was immobilized in the chitosan membrane, in which factors, such as the glutaraldehyde concentration, time of crosslinking and immobilization, buffer pH, were optimized with orthogonal experiments. The concentration of glutaraldehyde was finally chosen as 0.15%, time of crosslinking as 60 min, immobilization time as 15 h, and buffer PH as 8.0. The immobilization efficiency could reach up to 5U•cm2. The homemade microchip could be applied for rapid determination of glucose in serum, combining with the Luminol-K3Fe(SCN)3 Chemiluminescence reaction system. Under optimized conditions of sample velocity of 500uL•min-1, the concentration of K3Fe(SCN)3 of 1×10-2mol•L-1 and the concentration of luminol of 5×10-4 mol•L-1, the detection limit could achieved 1ug•mL-1.