Immobilized enzymes have drawn widespread attention due to the enhanced stability, easy separation from reaction mixture, and the prominent recyclability. Nevertheless, it is still an ongoing challenge to develop potent immobilization techniques which are capable of stable enzyme encapsulation, minimal loss of activity, and modulability for various enzymes and applications. Here, microfibers with tunable size and composition were fabricated using a home-made microfluidic device. These microfibers were able to efficiently encapsulate bovine serum albumin (BSA), glucose oxidase (GOX) and horseradish peroxidase (HRP). But the physically adsorbed enzymes readily diffused from microfibers into the catalytic reaction system. The leakage of enzymes could be substantially inhibited by conjugating to polyacrylic acid (PAA) and incorporating into the alginate-based microfibers, enabling stable immobilization, improved recyclability, and enhanced thermostability. In addition, GOX and HRP-loaded microfibers were fabricated under the optimized conditions for the visual detection of glucose using the cascade reaction of these enzymes, showing sensitive color change to glucose with concentration range of 0-2 mM. Due to the tunability and versatility, this microfluidic-based microfiber platform may provide a valuable approach to the enzyme immobilization for the cascade catalysis and diagnoses with multiple clinical markers.