A novel and versatile processing method was developed for the formation of nanoporous scaffold with in-situ enzyme immobilization for efficient biosensor applications. Our new approach used microfluidic devices to facilitate the single throughput, in-situ process to immobilize enzyme in a nanoporous scaffold via flow induced gelation, under ambient conditions. The nanoporous gel serves as a favorable host matrix for the immobilization of horseradish peroxidase (HRP) and ferrocene methanol (FcMeOH). The biosensor designed by the nanoporous scaffold demonstrated a highly linear amperometric response over the 0.1-1.5 mM range of H2O2 examined, with high sensitivity of 2.5 μM, high stability and selectivity, and good precision (RSD = 2.4%). This flow induced immobilziation technique opens up new pathways for designing simple, fast, biocompatible, and cost-effective process for enhanced sensor performance and on-site testing of a variety of biomolecules.