We developed a membraneless-microfluidic biofuel cell (MBFC) for the quantification of ethanol. The system employs anolyte and catholyte solutions, each containing a biocatalyst and redox mediator. The laminar flow conditions in the microfluidic chip minimize the mixing between anolyte and catholyte and obviate the need for a membrane to separate them. When ethanol is added to the anolyte, alcohol dehydrogenase (ADH) catalyzes its oxidation to acetaldehyde, releasing electrons to the anode. On the cathode, electrons are transferred to horseradish peroxidase (HRP), which reduces hydrogen peroxide in the catholyte to water. We optimized key design factors and operating conditions. We also studied the incorporation of glycerol as a viscosity modifier, which improved the power and current density supplied by the MBFC, with a maximum power output of 307 µW cm