In this work, the performance and mechanism for the boosting effects of fructose as an additional co-metabolite towards the biological treatment of reactive black 5 were systematically investigated. A decolorization efficiency of 98% was obtained in sample FRU200 (with 3 g/L fructose added based on 3 g/L yeast extract), which was 21% higher than that without fructose. Several intermediates with low molecular weight generated in sample FRU200 and different metabolic pathways were deduced. The bacterial community structure significantly changed due to fructose addition. Label-free quantitative proteomic approach suggested that several up-regulated proteins in sample FRU200 might play essential roles during the degradation. Furthermore, the mechanisms of 13.135 degradation by proteins/enzymes of the dominant species in flora DDMZ1 were proposed. This work deepens our understanding of the molecular and ecological mechanism of fructose as co-metabolite enhancing the biodegradation of refractory organic pollutants by a natural bacterial flora.