We report studies into the effects of electrolyte membrane characteristics on power generation in glucose fuel cells. Commercially available perfluo-rosulfonate ionomer membranes of various thicknesses were characterized by quantitatively measuring and analyzing electrical power generation. Characterization suggested that thinner Nafion® membranes could lead to improved performance (higher peak power, lower ohmic loss, and lower activation loss) over thicker membranes, however extremely thin membranes gave low performance due to their lack of stability, stemming from fabrication failure. The dependency on fuel concentration was also identified and a glucose concentration of 1 M gave the best performance. Finally, the prepared ion-exchange polymer composite membrane (formed by sorbing a Nafion® membrane into a polycarbonate track-etched (PCTE) membrane) was tested, and it was observed that cell performance was remarkably improved when membrane porosity was considered quantitatively. Our study is expected to be useful in the design of biofuel cells that can be utilized in low-concentration environments, such as living or-ganisms.