Paper-based enzyme immobilization for flow injection electrochemical biosensor integrated with reagent-loaded cartridge towards portable device was developed.A paper disk was immobilized with enzyme,then it was integrated in a flow cell as an electrochemical biosensor.Silicon tube reagent-loaded cartridge was integrated into the system,complicated procedure was simplified as one-click operation towards development for point-of-care applications.In this research,glucose oxidase (GOx) was employed as a model enzyme,silver ion as an inhibition reagent for GOx,EDTA as a regeneration reagent.When GOx was inhibited by silver ions,glucose was introduced for electrochemical measurements before and after inhibited enzyme regeneration,the difference was caused by silver inhibition.The modular device has great potential for other applications,e.g.detection of enzyme activity and substrate.The platform based on double-test mode provided accurate results due to elimination of an average or control value in comparison with routine approaches.