This paper describes the design and testing of a microfluidic biofuel cell that uses a flow-through bioanode and an air-breathing cathode. The bioanode is Toray carbon paper with glucose dehydrogenase (GDH), multi-walled carbon nanotubes (MWCNTs), and methylene green immobilized within a hydrogel. The cathode consists of a commercially available air-breathing platinum cathode hot pressed to a Nafion membrane. All remaining biofuel cell components were laser-cut from poly(methyl methacrylate) (PMMA) and silicone sheets. Half-cell experiments indicate that cathode variability limits the biofuel cell. An examination of flow rate effects on the biofuel cell showed that the current density increased sharply up to about 1 mL/min. Tested at this flow rate, the flow-through biofuel cell achieved a maximum current and power density of 705 mu A/cm(2) and 146 mu W/cm(2). This was a 6% and 29% improvement in the current and power density, respectively, compared to the previously demonstrated bioanode without flow. Fuel utilization was calculated based on the measured current and by measuring UV-Vis absorbance of the reduced form of hydroxybenzhydrazide. The maximum fuel utilization was 5.8% at a flow rate of 0.05 mL/min. Finally, a numerical model of the biofuel cell was designed and its results compare favorably to actual data. (C) 2013 The Electrochemical Society. All rights reserved.