Microbial fuel cells (MFCs) have attracted considerable attention as potential biosensors. A MFC biosensor for rapid measurement of biochemical oxygen demand (BOD) has been recently studied. However, a standardized bacterial mixture inoculated in the MFC biosensor for BOD measurement is unavailable. Thus, the commercial application of a MFC biosensor is limited. In this study, a mediator-less MFC biosensor inoculated with known mixed cultures to quickly determine BOD concentration was tested. Optimal external resistance, operating temperature and measurement time for the MFC biosensor were determined to be 5000 Ω, 35°C and 12 h, respectively. A good relationship between BOD concentration and voltage output, high reproducibility and long-term stability for the MFC biosensor was observed. The newly developed MFC biosensor was inoculated with a mixture of six bacterial strains (Thermincola carboxydiphila, Pseudomonas aeruginosa, Ochrobactrum intermedium, Shewanella frigidimarina, Citrobacter freundii and Clostridium acetobutylicum) capable of degrading complex organic compounds and surviving toxic conditions. The described MFC biosensor was able to successfully measure BOD concentrations below 240 mg L−1 in real wastewater samples.