Viamin K₃-modified poly-l-lysine (PLL-VK₃) was synthesized and used as the electron transfer mediator during catalytic oxidation of NADH by diaphorase (Dp) at the anode of biofuel cell. PLL-VK₃ and Dp were co-immobilized on an electrode and then coated with NAD⁺-dependent glucose dehydrogenase (GDH). The resulting enzymatic bilayer (abbreviated PLL-VK₃/Dp/GDH) catalyzed glucose oxidation. Addition of carbon black (Ketjenblack, KB) into the bilayer enlarged the effective surface area of the electrode and consequentially increased the catalytic activity. An oxidation current of ca. 2mAcm⁻² was observed when the electrochemical cell contained a stirred 30mM glucose, 1.0mM NAD⁺, pH 7.0 phosphate-buffered electrolyte solution. The performance of glucose/O₂ biofuel cells, constructed as fluidic chips with controllable fuel flow and containing a KB/PLL-VK₃/Dp/GDH-coated anode and an Ag/AgCl or a polydimethylsiloxane-coated Pt cathode, were evaluated. The open circuit voltage of the cell with the PDMS-coated Pt cathode was 0.55V and its maximum power density was 32μWcm⁻² at 0.29V when a pH 7.0-buffered fuel containing 5.0mM glucose and 1.0mM NAD⁺ was introduced into the cell at a flow rate of 1.0mLmin⁻¹. The cell's output increased as the flow rate increased. During 18h of continuous operation of the cell with a load of 100kΩ, the output current density declined by ca. 50%, probably due to swelling of the enzyme bilayer.