The objective of the present research was to explore new approach to reduce the hydrolysis time and to enhance the productivity of enzymatic saccharification. One-stage hydrolysis of steam-exploded corn stover required 72 h to reach a yield of 62.8%, while multi-stage hydrolysis could reduce the time to 24 h. A concept of three-stage hydrolysis was therefore proposed in which cellulosic substrate was hydrolyzed for 6, 6, and 12 h, respectively. High hydrolysis yields, 70.2% with enzyme recycling and 76.1% with the supplement of fresh enzyme to eliminate enzyme recovery procedure, were obtained in 24 h. Analysis indicated that short-time hydrolysis and the removal of end products at each stage improved cellulase activities and benefited the adsorption of cellulase enzyme to the solid substrate. When steam-exploded corn stover was used as the substrate for cellulase synthesis, a hydrolysis yield of 88.6% was achieved in 24 h. (C) 2009 Elsevier Ltd. All rights reserved.