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We report a μL-scale microfluidic Microbial Fuel Cell (MFC) having optimal biofilm formation and minimal oxygen invasion into its anode chamber to generate high power density. The maximum power density of our μL-scale MFC is 95 μW/cm2, the highest value among previously reported μL-scale MFCs and even comparable to that of macro-scale counterparts. The biofilm formed by exoelectrogens, G. sulfurreducens, was studied by using four different thicknesses of photo-definable polydimethylsiloxane (PDMS) spacer; 10, 20, 55, and 155 μm. Both current and power densities are significantly limited when the PDMS spacer is less than 55 μm thick.