Microbial fuel cells (MFCs) were embedded into constructed wetlands to form microbial fuel cell coupled constructed wetlands (CW-MFCs) and were used for simultaneous azo dye wastewater treatment and bioelectricity generation. For the first time, the effects of different substrate biomass on the performance of CW-MFCs were studied. Group A had the highest substrate biomass of 0.453 g VSS per L, followed in order by group D, group B and group C. CW-MFCs with more substrate biomass showed higher decolorization efficiencies but lower electrode performance. The decolorization efficiency and the maximal power density of group A were 92.7% and 0.117 W m⁻³, respectively, while the decolorization efficiency and the maximal power density of group C were 76.26% and 0.256 W m⁻³, respectively. The cathode performance had the greatest impact on the CW-MFC performance. This may be due to the high cathode activation resistance which may be caused by the lack of cathode microbes. The substrate biomass exerted significant effects on the electrode microbes. The CW-MFCs with more substrate biomass had fewer electrode microbes, which may reduce the electrode performance. This study highlights the applications of CW-MFCs and other MFCs that were built in a natural environment.