In this study, batch biohydrogen production by co-digestion of raw rice straw and activated sewage sludge was investigated with different inoculum heat treatment, pH, S/X ratio (based on VS) and substrate sizes under mesophilic condition. In order to achieve a high bio -hydrogen yield and methanogens activity inhibition, heat treatment of inoculum was optimized at different exposure times (30, 45 & 60 min) and temperature ranges (80, 90 and 100 degrees C) prior to dark fermentation process. Collected data was analysed using response surface methodology (RSM). The heat treatment of inoculum at 100 degrees C for 60 min produced the highest bio-hydrogen yield of 14.22 NmL H-2/g VS at concentration of 70.97% and Production of 0.073 NmL CH4/g VS at 0.17% concentration in total produced biogas. The raw rice straw was also co-digested with heat-treated inoculum at different ratios of volatile solids (2:1, 4:1 and 6:1) and initial pH (4, 4.75 and 5.5) as numerical variables and 4 categories of substrate size ((250-500 mu m], (500 mu m-2mm], (2-20 mm), [20-30 mm]). The highest bio-hydrogen yield of 14.70 NmL/g VS was recognized at the optimum initial pH of 5.01 and S/X ratio of 4.54:1 using 2-20 mm rice straw. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.