This paper deals with the preparation and characterization of low-cost sulfonated polystyrene (SPS) proton exchange membrane (PEM) as well as its application in a double-chambered microbial fuel cell by using glucose solution as a substrate. Characterization of membrane has been done by diffraction scanning calorimetry (DSC), thermogravimetric analysis (TGA), energy-dispersive X-ray spectroscopy (EDX), field emission scanning electron microscopy (FESEM) (before and after the use of 1100 h), Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (HNMR), and few other methods. Performance of SPS membrane is studied in microbial fuel cell (MFC) in terms of voltage, power density generated, and organic material removal with variable process parameter initial chemical oxygen demand (COD) concentration, initial pH, and temperature. Box-Behnken model with three factors (initial COD, initial pH, and temperature) and three levels is used to fix the experimental conditions for optimization of voltage and current density using Design Expert software. Maximum voltage and power density generation of 647 mV and 104.7 W/m3 have been obtained at initial COD 1500 mg/L, anodic pH 7, and temperature 35 °C.