This study compares the performance of different pretreatment and hydrolysis methods for the efficient production of fermentable sugars from rice mill wastewater. The pretreatment and hydrolysis operating conditions were optimized. The maximum reducing sugar concentration of 16.1 g/L was obtained with combined acid and enzymatic hydrolysis. The heat, alkali, ultrasound pretreatment, acid hydrolysis and enzymatic hydrolysis showed 2.9, 2.7, 2.1, 14.2 and 15.8 g/L reducing sugar concentration, respectively. The hydrogen production was carried out using Enterobacter aerogenes RM 08 with hydrolyzate obtained from combined acid and enzymatic hydrolysis and the results showed 1.97 mol H-2/mol of sugar. The cumulative hydrogen production and the growth data of E. aerogenes RM 08 were fitted with modified Gompertz and Logistic equations. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.