Synthesis and characterization of Nafion/TiO2 membranes (TiO2 1 wt%) with different solvents (DMF, DMAc, NMP) for proton exchange membrane operating at Microbial Fuel Cell (MFC) was investigated in this study. Nanocomposite membranes are studied due to their better physical properties and higher production voltage in comparison with Nafion 112 in MFC systems. Nafion/TiO2 nanocomposite membranes were prepared by solution casting Method. The structures of membranes were investigated by Scanning Electron Microscopy (SEM). In addition, water uptake, proton conductivity, and ion exchange capacity (IEC) of membranes were measured and compared with Nafion 112 in microbial fuel cell. The nanocomposite membrane prepared with DMF solvent has the best morphology, the highest porosity, and the maximum proton conductivity. Subsequently, the membranes were tested in a real MFC system and the nanocomposite membrane prepared with DMF solvent exhibited the maximum MFC voltage. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.