A novel, facile and green approach for the fabrication of H2O2, glutathione (GSH) and glucose detection biosensor using water-stable PVA and PVA/PEI nanofibers decorated with AgNPs by combining an in situ reduction approach and electrospinning technique has been demonstrated. Small, uniform and well-dispersed AgNPs embedded in the PVA nanofibers and immobilized on functionalized PVA/PEI nanofibers indicate the highly sensitive detection of H2O2 with a detection limit of 5 μM and exhibit a fast response, broad linear range, low detection limit and excellent stability and reusability.