Abstract: The layer-by-layer structured composite film of graphene-Prussian blue (PB)-chitosan (CS) was fabricated on a glassy carbon electrode by three-step electrodeposition method and used for glucose sensing. Graphene nanosheets were directly deposited onto the electrode through reduction of graphene oxide by cyclic potential scanning. Then, a glucose biosensor was fabricated by electrodepositing PB nanoparticles and glucose oxidase (GOD)-chitosan hybrid film (GOD-CS) on the graphene modified electrode successively. The surface of the resulting modified electrode was characterized by electrochemical methods and scanning electron microscopy. Under optimal conditions, the biosensor shows high sensitivity (50.29 mA· L·mol-1·cm-2), low detection limit (12 μmol·L-1) and fast response time (3 s). A linear dependence of the catalytic current upon glucose concentration is obtained in a wide range from 0.02 to 10 mmol·L-1. In addition, the sensor also performs well for measuring glucose concentrations in human blood serum samples without any pretreatment.