High chemical input is required for enzymatic production of xylo-oligosaccharides (XOS) using xylan extracted from lignocellulosic biomass. In this study, enzymatic hydrolysis of alkaline oxidation (AO) treated sugarcane bagasse (SCB) directly for the production of XOS was conducted. The effect of AO pretreatment on the chemical compositions and hydrolytic properties of SCB was investigated. The AO pretreatment conditions with low chemical input for the production of XOS were optimized by orthogonal design. Stepwise enzymatic hydrolysis of AO pretreated SCB with xylanase and cellulase produced XOS (1.78 g/L), meanwhile, the cellulose conversion increased from 84.97% to 91.51% compared with directly enzymatic hydrolysis using cellulase. HPLC-UV and MALDI-TOF-MS analysis indicated that the obtained XOS products were mainly composed of xylobiose and xylose with a small amount of arabinose/4-O-methylglucuronic acid substituted xylotriose and xylotetraose. The proposed strategy for the co-production of functional XOS and fermentable sugars from SCB showed potential of industrial application.