An effective way to prepare the reagentless electrochemical microbial biosensor has been developed. First, the mediator thionine (TH) is wrapped on the surface of E. coli (E. coli@TH) through the simple electrostatic interaction, which makes E. coil and TH closely together and therefore shortens the electron transfer distance between the electroactive centers and the electrode. Then, E. coli@TH together with chitosan (CHI)-entrapped carbon nanodots (CNDs)are immobilized onto the glassy carbon electrode (GCE) surfaces to form the reagentless electrochemical microbial biosensor (CHI-CNDs-E. coli@TH), in which the CNDs are used to improve the conductivity of the modified electrode, and further enhance the electron transfer from E. coli to the electrode. The reagentless CHI-CNDs-E. coli@TH biosensor is finally used to evaluate the toxicity of four heavy metal ions (Cd2+, Cu2+, Pb2+, Zn2+) and three real wastewater samples, showing good performance in the evaluation of wastewater toxicity compared to other reported methods. This work opens up a new way for the preparation of reagentless electrodes, making it possible for electrochemical microbial biosensors to be equipped and realize on-line detection. (C) 2016 Elsevier Ltd. All rights reserved.