The utilization of ultrasonic treatment on digestion sludge to enhance microbial activity for bio-hydrogen production was investigated. The optimal conditions of ultrasonic time and density on digestion sludge were detected using Central Composite Experimental Design. The regression analysis showed that a significant increase of 1.34 fold in bio-hydrogen production rate could be obtained when ultrasonic time was 10 s and ultrasonic density around 130 W/l at digester sludge concentration of 15 g VSS/l. The analyses of biodegradation characteristics in bio-hydrogen producing process implied that ultrasound did not denature the digestion sludge but just improved its biodegradation efficiency. In order to find out the mechanism of ultrasonic treatment on digestion sludge, a control experiment was designed and COD values of digestion sludge in different treatment conditions was measured. Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu.