A combined deacetylation and PFI refining pretreatment was applied to corn cob for the improvement of a two-stage enzymatic hydrolysis. In stage 1, the pretreated corn cob was first hydrolyzed by xylanase to produce xylo-oligosaccharides (XOS). In stage 2, the solid residue isolated from stage 1 was further hydrolyzed by cellulase and beta-glucosidase. NaOH, Na2CO3, and Ca(OH)(2) were tested to remove acetyl groups in the process of deacetylation, and it was found that Ca(OH)(2) could be the most suitable alkali for deacetylation in this work. After deacetylation using 0.8 mmol of Ca(OH)(2)/g of substrate and PFI refining, 50.5% xylan in the raw material could be hydrolyzed into XOS. The corresponding xylan yield of stage 1, the glucan yield of stage 2, and the total sugar yield (all sugars released in the hydrolyzate) after the two-stage enzymatic hydrolysis were 0.306, 0.305, and 0.661 g/g of corn cob, respectively.