Xylo-oligosaccharides, as the important intermediates of hemicellulose degradation, widely exist in hydrolysate from pretreatment and enzymatic hydrolysis of lignocellulose. This study showed that xylo-oligosaccharides largely reduced the efficiency of cellulose hydrolysis, but this inhibition cannot be effectively relieved by increasing loading of cellulose substrate or cellulase. Xylo-oligosaccharides largely suppressed enzymatic activities of cellobiohydrolases, endoglucanases, and beta-glucosidase of cellulase complexes, especially cellobiohydrolases, and adding cellobiohydrolases or suitable beta-glucosidase to hydrolysis systems could increase cellulose conversion. Thin-layer chromatography assay indicated that xylotetraose was quickly degraded in the initial hydrolysis stage while xylotriose was still held a higher concentration until 24 h of hydrolysis, and it is speculated that xylotriose was a stubborn inhibitor of enzymatic hydrolysis. The molecular docking analysis showed that xylotriose preferred to combine in the tunnel of cellobiohydrolases, which was generally recognized as the substrate-binding site on cellobiohydrolases, and thus reduced the effectiveness of cellobiohydrolases.