Abstract Microfluidic enzyme-based reactor could be used to detect biomolecules with high sensitivity and selectivity. Based on the electrophoretic-mediated microanalysis (EMMA) method, a novel free enzyme-based microchip was developed to detect glucose. Online catalysis reaction of glucose by glucose oxidase was carried out on the chip. The product H 2 O 2 was detected using amperometric method. The working electrode, single carbon fibre cylindrical electrode, was held at a constant potential of +1.4 V versus Ag/AgCl electrode. The results indicated that the peak current had a good linear relationship with glucose concentration in the range of 0.1–20 mM ( R = 0.997) with good reproducibility (RSD = 2.02%, n = 10). The detection limit of glucose was 19.8 μM ( S/N = 3). This free enzyme-based reactor was easy to fabricate and operate, which showed great potential application in bioanalysis.