The current study was made to develop a biosensor based on a single-chamber microbial fuel cell in which anaerobes were retained in the anode compartment separated from the cathode compartment by a proton exchange membrane. In the sensor a replaceable anaerobic consortium was used for analyzing biodegradable organic matter. The anaerobes acted as biocatalysts in oxidizing organic matter and transferring electrons to the anode. The biocatalysts were renewed for each sample analysis by replacing the old anaerobic consortium with an equal amount of fresh one. A glucose standard solution was used as the target substrate. To obtain the maximum sensor output, the MFC-based sensor system was optimized using an 800 Ω resistor as the load to the external electric circuit and 25 mM phosphate buffer with 50 mM NaCl as catholyte in the aerobic compartment. The temperature of anaerobic compartment was maintained at optimal 37 °C. The cell potential across the electrodes increased with increasing loading of glucose. The sensor response was linear against concentration of glucose up to 25 g l−1. The detection limit was found as 0.025 g l−1. The microbial fuel cell with replaceable anaerobic consortium could be used as a biosensor for on-line monitoring of organic matter.