Azo dyes are toxic and carcinogenic and are often present in industrial effluents. In this research, azoreductase and glucose 1-dehydrogenase were coupled for both continuous generation of the cofactor NADH and azo dye removal. The results show that 85% maximum relative activity of azoreductase in an integrated enzyme system was obtained at the conditions: 1 U azoreductase:10 U glucose 1-dehydrogenase, 250 mM glucose, 1.0 mM NAD+ and 150 lM methyl red. Sensitivity analysis of the factors in the enzyme system affecting dye removal examined by an artificial neural network model shows that the relative importance of enzyme ratio between azoreductase and glucose 1-dehydrogenase was 22%, followed by dye concentration (27%), NAD+ concentration (23%) and glucose concentration (22%), indicating none of the variables could be ignored in the enzyme system. Batch results show that the enzyme system has application potential for dye removal.