Compared with free enzymes, immobilized enzymes have improved stable high enzymatic efficiency and can be reused and separated easily. In this study, glucose oxidase (GOx) was immobilized in the inlet end of capillary as a plug and an online immobilized GOx capillary enzyme microreactor was developed. The enzymatic hydrolysis, electrophoretic separation and detection could be accomplished simultaneously via the prepared capillary, which combined the advantages of both enzyme microreactor and capillary electrophoresis. The present approach has potential applications in high-throughput screening of enzyme reactions and enzyme inhibitors.