Xylo-oligosaccharides (XOS) with prebiotic properties are obtained through xylan hydrolysis. Previously organosolv was found to be an effective pretreatment for XOS production from corncob. However, this process suffered from low XOS yields because of the limited hydrolysis of xylan in the pretreated biomass. This study was designed to test some approaches toward increasing the efficiency of xylanases on the organosolv-treated corncob. The two commercial enzymes (Shearzyme 500L and Veron 191S) used in this study showed a synergistic effect yielding higher XOS compared to single enzyme application. Partial removal of acetyl groups on the xylan in organosolv-treated corncobs enhanced XOS production significantly. The solid loading above 10% decreased XOS yield as it resulted in a highly viscous slurry that may have limited heat and mass transfer. Fed-batch addition of enzyme and biomass did not play a role in the improvement of hydrolysis. In sequential batch mode, the residual enzyme activity from the previous batch could release more XOS from the fresh biomass, though the yield was low. Addition of fresh enzymes to the previous hydrolysate together with biomass provided a more concentrated XOS solution (15.4 g/l) after the second batch. The addition of surfactants into the hydrolysis media to prevent enzyme binding to lignin did not improve XOS production. This study showed that organosolv could be considered an effective treatment for XOS production from corncob and the enzymatic hydrolysis could be improved by optimizing the conditions.