In this study, a novel, single-chamber bioelectrochemical system (BES) with stacked modules was developed to improve azo dye decolorization. The decolorization extent of BES with three modules (80.3 +/- 3.1%) was about 15% and 33% higher than that with two modules (65.6 +/- 4.5%) and one module (47.1 +/- 3.9%) at an influent Acid Orange 7 (AO7) loading rate of 250 gm(-3) d(-1), demonstrating the feasibility of enhanced decolorization in scale-up BES through increasing the number of stacked modules. Moreover, the contribution of each module to dye decolorization, chemical oxygen demand (COD) removal and volatile fatty acids (VFAs) changes, showed the sequential utilization of pollutants in the stacked BES. Furthermore, analysis of the microbial communities showed that Pseudomonas, Geobacter, Comamonas, Meniscus, Bellilinea, Achromobacter, and Paludibacter were significantly enriched and resulted in the specific microbial community structures of the biocathode, meanwhile Geobacter and Acinetobacter were enriched in the microbial community of the bioanode, which mainly contributed to the electron transfer and/or azo dye decolorization. The results of this study demonstrate the potential of stacked BES for the accelerated deployment of large-scale BES applications for the treatment of refractory waste streams such as azo dye-bearing wastewater.