One of the technologies considered for bioenergy production is microbial fuel cell. The microbial fuel cells are used as a novel method for wastewater treatment and power generation simultaneously. In this method, microorganisms appear as catalysts to convert chemical energy stored in organic matter into electrical energy under anaerobic conditions. In this study, a microbial fuel cell was designed and constructed using microorganisms existing in municipal wastewater to generate electricity. The structure of the current microbial fuel cell was single-chamber, into which added the wastewater. This chamber was adjacent to the anode and fed with anaerobic sludge, existing in municipal wastewater. In the constructed fuel cell, an air-cathode was used which was separated from the anode chamber by a proton exchange membrane. In order to measure voltage at different currents, a variable resistance and a digital multimeter with data storage capability were used. In this study, the developed potential differences due to changes in the type of microorganism, substrate, and the value of external resistance were investigated. Among the two types of substrates molasses and industrial vinasse investigated, industrial vinasse produced more voltage. The maximum current density of 312.7843mA/m2 was measured at 200 Ohm resistance and maximum power density at 600 Ohm resistance and current density of 201.41mA/m2 was measured 85.6010mW/m2.