A simple and efficient method using enzyme immobilized microfluidic channel as open tubular microreactor was designed for amperometric detection of glucose. The microreactor was composed of a polydimethylsilicone/glass hybrid device with three reservoirs, a cooling cave and a 6 cm capillary with a sampling fracture as microchannel. The microchannel was further modified by thermal polymerization, followed by covalently attaching with glucose oxidase. Through fracture sampling and electrochromatography separation, the production via enzymatic reaction was determinated by Pt electrode at the end of capillary. The linear range for the detection of glucose was 0.05–7.5 mmol·L−1 with detection limit of 23 µmol·L−1. The inter- and intra-chip reproducibilities for determination of 2.5 mmol·L−1 glucose were 98.5% (n=5) and 96.0% (n=5), respectively. With the advantage of flexible assembly, rapid efficiency, good stability and low-cost, this microreactor provided a potential platform for establishing a portable enzyme-based chemical detection system in practical application.