The textile industry is developing rapidly in China. It generates large volumes of cotton dyeing pretreatment wastewater (CDPW). CDPW contains high concentrations of pollutants characterized by their strongly alkaline and recalcitrant nature for microbial degradation. This project aimed to evaluate the performance of a microbial electrolysis cell (MEC) coupled with anoxic/oxic (A/O) system (MEC-A/O) in treating CDPW, as well as analyze changes in microbial diversity. The results indicated that the effect of biological treatment in an electrolytic cell to treat CDPW was optimal at the voltage of 0.6V. The chemical oxygen demand (COD) removal efficiency under optimum conditions was 69.13%, higher than that of the A/O system alone (48.93%). Within a certain range, applied voltage was able to enhance microbial activity, increase the sludge concentration and enlarge the sludge particle size. At the same time, the applied voltage could effectively increase the abundance and the diversity of Bacteria and Archaea, as well as accelerate the degradation of pollutants.