The present work describes an effectiveness of ultrasonication pretreatment for enhancement of biohydrogen production from complex dairy wastewater at optimized culture conditions obtained previously. An ultrasonic pretreatment was comprised of five ultrasonic densities (UD) (from 0 to 0.2 W/mL) at five different ultrasonication time (UT) (from 6 to 14 min). Response surface methodology (RSM) analysis showed that UD was the highly significant parameter, which predominantly influences biodegradability, hydrogen yield (HY), and specific hydrogen production rate (SHPR) enhancement during dark fermentation. Optimization of pretreatment using RSM integrated with desirability function followed by a verification test showed that maximum HY of 15.33 mmol/g COD and SHPR of 31.38 mmol/g VSS.d obtained at 0.08 W/mL and 9 min. Significant relative enhancement of HY, 27%, and SHPR, 51%, suggests that ultrasonication pretreatment of complex dairy wastewater is about 1.1-2-fold more effective for enhanced hydrogen recovery compared to unsonicated one. A successful energy balance of ultrasonication pretreatment suggests an effectiveness of the pretreatment for enhanced energy recovery. (C) Copyright 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.