Since most risk assessment for toxicants is based on individual single-species test, the deduction of such results to ecosystem evaluation is afflicted with uncertainties. Herein, we successfully developed a p-benzoquinone mediated whole-cell electrochemical biosensor for multi-pollutants toxicological analysis by co-immobilizing mixed strains of microorganism, including Escherichia coli (gram-negative bacteria), Bacillus subtilis(gram-positive bacteria) and Saccharomyces cerevisiae (fungus). The individual and combined toxicities of heavy metal ions (Cu²⁺, Cd²⁺), phenol (3,5-dichlorophenol) and pesticides (Ametryn, Acephate) were examined. The experimental results showed that the order of toxicity for individual toxicant was ranked as Cu²⁺ > 3,5-dichlorophenol (DCP) > Ametryn > Cd²⁺ > Acephate. Then the toxic unit (TU) model was applied to determine the nature of toxicological interaction of the toxicants which can be classified as concentration additive (IC50_mix = 1TU), synergistic (IC50_mix < 1TU) and antagonistic (IC50_mix > 1TU) responses. The binary combination of Cu²⁺ + Cd²⁺, Cu²⁺ + DCP, Cu²⁺ + Acephate, DCP + Acephate, Acephate + Ametryn were analyzed and the three kind of joint toxicity effects (i.e. additive, synergistic and antagonistic) mentioned above were observed according to the dose-response relationship. The results indicate that the whole-cell electrochemical biosensor based on mixed microbial consortium is more reasonable to reflect the joint biotoxicity of multi-pollutants existing in real wastewater, and combined effects of toxicants is extremely necessary to be taken into account in ecological risk assessment. Thus, present study has provided a promising approach to the quality assessment of wastewater and a reliable way for early risk warning of acute biotoxicity.