Dark fermentation of distillery wastewater (DWW) gives a lower hydrogen yield (HY) and hydrogen production rate (R), owing to the complexity and a recalcitrant nature of effluent. Therefore, an effective pretreatment of DWW becomes imperative for the improvement of biohydrogen production. In the present study, the efficacy of ultrasonic pretreatment for enhancement of biohydrogen production from DWW was evaluated in batch test. Several variables, such as COD input, ultrasonic density (UD), and ultrasonication time (UT) were studied for optimization using response surface methodology integrated with desirability function. The highest HY, 10.95 mmol/g COD, and R, 6.67 mmol/L h, were obtained for batch test of ultrasonically pretreated DWW under optimal conditions for COD, UD and UT at 56 g/L, 0.12 W/mL, and 17 mm, respectively. The significant relative enhancement of HY, 101%, and R, 103%, implies that ultrasonically treated DWW is about 1.2-1.4 times more effective for enhanced biohydrogen production from complex DWW compared to unsonicated DWW. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.