In this work, a simplified procedure of detection of water toxicity based on Pt ultramicroelectrode (UME) and mixed microorganism cultured without sterilization was the first proposed. A stable Pt UME was successfully prepared with a special glass tube as insulation and support material, which was used as working electrode in the biosensor. The Pt UME exhibits the typical cyclic voltammogram (CV) of Pt UME with sigmoid shape and possesses good stability, enlarged current response and tunable dimension. In addition, it was an effective and simple method for toxicity biosensor using mixed microorganisms cultured in unsterilized lysogeny broth (LB) as the bioreceptor. K-3[Fe(CN)(6)] was used as an electron mediator. Under the optimal conditions of 30 mM K-3[Fe(CN)(6)], OD600 = 1 cell concentration, and 50 mM phosphate-buffered solution (PBS), the half-maximal inhibitory concentration (IC50) values measured for Cd2+, Cu2+ and Ni2+ were 3.99 mg/L, 1.16 mg/L and 2.37 mg/L, respectively. The results indicated that the biosensor with large diameter Pt UME and mixed microorganisms cultured in unsterilized LB realized rapid and simple detection of water toxicity.