Waste pastry was hydrolyzed by glucoamylase and protease which were obtained from solid state fermentation of Aspergillus awamori and Aspergillus oryzae to produce waste pastry hydrolysate. Then, the effects of hydraulic retention times (HRTs) (4-12 h) on hydrogen production rate (HPR) in the suspended microbial growth system (continuous stirred tank reactor, CSTR) and attached microbial growth system (continuous mixed immobilized sludge reactor, CMISR) from waste pastry hydrolysate were investigated. The maximum HPRs of CSTR (201.8 mL/(h.L)) and CMISR (255.3 mL/(h.L)) were obtained at HRT of 6 h and 4 h, respectively. The first-order reaction could be used to describe the enzymatic hydrolysis of waste pastry. The carbon content of the waste pastry remained 22.8% in the undigested waste pastry and consumed 77.2% for carbon dioxide and soluble microbial products. To our knowledge, this is the first study which reports biohydrogen production from waste pastry. (C) 2016 Elsevier Ltd. All rights reserved.