Lignocellulosic biomass, if properly saccharified, could be an ideal feedstock for biohydrogen production. However, the high cellulases cost is the key obstacle to its development. In this work, cost-effective enzyme produced by Trichoderma viride was used to saccharify cornstalk. To obtain high sugar yield, a central composite design of response surface method was used to optimize enzymatic saccharification process. Experimental results showed that the enzymatic saccharification rate reached the highest of 81.2% when pH, temperature, cellulases and substrate concentration were 5, 49.7 °C, 35.7 IU g−1, and 38.5 g L−1, respectively. The cornstalk hydrolysate was subsequently introduced to fermentation by Thermoanaerobacterium thermosaccharolyticum W16, the yield of hydrogen reached the highest level of 90.6 ml H2 g−1 pretreated cornstalk. The present results indicate the potential of using T. thermosaccharolyticum W16 for high yield conversion of cornstalk hydrolysate, which was saccharified by onsite enzyme produced by T. viride.