Microfiltration membrane, a potential alternative for traditional proton exchange membrane (PEM) due to its strong ability of proton transfer, cost-effectiveness, sustainability and high anti -pollution capability in microbial fuel cell (MFC). In this study, a novel MFC using bilayer microfiltration membrane as separator, inoculated sludge as biocatalyst and P-nitroaniline (PNA) as electron donor was successfully constructed to evaluate its performance. Furthermore, we also investigated the effects of initial PNA concentration, co-substrate (acetate) and cultivated microorganisms on MFC performance. Results showed that the maximum power density of 4.43, 3.05, 2.62 and 2.18 mW m(-2) was acquired with 50, 100, 150 and 300 mg L-1 of PNA as substrate, respectively. However, with the addition of 500 mg L-1 of acetate into reaction system contained 100 mg of PNA, the higher power production of 6.24 mW m(-2) was obtained, which was 2.05 times higher than that using 100 mg L-1 of PNA as the sole substrate. Meanwhile, the MFC working on cultivated microorganisms displayed a maximal power density of 7.32 mW m(-2) and a maximum PNA degradation efficiency of 54.75%. And after an electricity production cycle, the number of microbes in the anode chamber significantly increased. This study provides a promising technology for bioelectricity generation by biodegrading biorefractory pollutants in wastewater. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.