A simple mediated microbial biosensor providing real-time monitoring of water quality and evaluation of biotoxicity was fabricated by entrapping Escherichia coil (E. coil) cells in gelatin on glassy carbon electrode with benzoquinone as the redox mediator. The biotoxicity assay was based on the respiratory activity of coil cells estimated by the oxidation current of microbially reduced benzoquinone. The neutrality and lipophilicity rendered benzoquinone better efficiency than ferricyanide in mediated microbial reactions. After the optimization of preparation conditions, the prepared microbial biosensors have measured several common toxicants with different concentrations. In addition, the biotoxicity of binary mixtures of heavy metals and wastewater were investigated. The fabricated biosensor exhibited good repeatability and stability in the biotoxicity measurements. (C) 2013 Elsevier Ltd. All rights reserved.