Starch is one of the most abundant resources on earth and is suited to serve as a cost-effective feedstock for biological hydrogen production. However, producing hydrogen from direct fermentation of starch is usually inefficient, as the starch hydrolysis is often the rate-limiting step. Therefore, in the present work, enzymatic starch hydrolysis was conducted to enhance the feasibility of using starch feedstock for H-2 production. The amylase (with a molecular weight of ca. 112 kDa) used for starch hydrolysis was produced from a recombinant E. coli harboring an amylase gene originating from Caldimonas taiwanensis On1. Using statistical experimental design, the optimal pH and temperature for starch hydrolysis with the recombinant amylase was pH 6.86 and 52.4 degrees C, respectively, at an initial starch concentration of 7 g/L. The hydrolyzed products contained mainly glucose, maltotriose, and maltotetrose, while a tiny amount of maltose was also detected. The enzymatically hydrolyzed products of soluble starch and cassava starch were used as the substrate for dark hydrogen fermentation using Clostridium butyricum CGS2 and, Clostridium pasteurianum CH4. The highest H-2 production rate (nu(H2)) and yield (Y-H2) of C. butyricum CGS2 was 124.0 mL/h/L and 6.32 mmol H-2/g COD, respectively, both obtained with the hydrolysate of cassava starch. The best H-2 production rate (63.0 mL/h/L) of C. pasteurianum CH4 occurred when using hydrolyzed cassava starch as the substrate, whereas the highest yield (9.95 mmol H-2/g COD) was obtained with the hydrolyzed soluble starch.