We introduce a bifunctional architecture to construct biosensors on solution-blow spinning fiber mats of polylactic acid (PLA) and polyethylene glycol (PEG). PLA/PEG mats acted as substrate for printing electrodes and as matrix to immobilize glucose oxidase (GOx). Prussian Blue nanoparticles (PB) decorated working electrodes to detect H₂O₂ from enzymatic catalysis at a low applied potential (0 V vs. Ag/AgCl) with detection limit of 0.197 mM. The inexpensive bifunctional device (<US $0.25 per unit) exhibited a rapid response, long lifetime, and performance similar to the standard method for glucose monitoring in human urine samples. The PLA/PEG mats are sustainable alternative for biosensing and wearable applications.