The aim of this study was to investigate the effect of the single and combined substrates, namely rice hulls and/or cotton stalks hydrolysates media prepared by dilute acid hydrolysis on the bioethanol production using Escherichia coil KO11 for the economic feasibility of efficient production system. The cells were incubated on an orbital shaker at a shaking frequency of 160 rpm under semi-anaerobic conditions at the temperature of 30 degrees C during 96 h of the fermentation period. The ethanol concentration reached the maximum level of 20.69 g/L with the maximum yield of 0.44 (g ethanol/g reducing sugar) when the biomass concentration was 2.32 g/L corresponding to a growth rate of 0.023 h(-1) at the highest C/N ratio (27.13) in the combination of 70% rice hulls and 30% cotton stalk hydrolysate medium at the end of the fermentation period of 96 h. The experimental results indicated that the combination of cellulosic substrates proves to have positive effects on the bioethanol production. (C) 2014 Elsevier Ltd. All rights reserved.