Abstract: Abstract: The utilization of microorganisms or their enzymes may be the most promising method to produce Xylo-oligosaccharides (XOs) from agricultural wastes because of fewer undesirable by-products and fewer monosaccharides produced, and no special equipments required. Industrial-scale production of XOs using bioconversion requires low-cost substrates with a high xylan content, low lignin content and enzymes with high activity, process stability and β-1,4-xylosidic bond speci?city. In the current study, bagasse, by-products of sugarcane production, was used as substrates for XOs production. And hydrolysis of bagasse hemicellulose was carried out with a recombinant multi-functional xylanase (RMFXase). RMFXase is a novel enzyme produced from genetically engineered straw mushroom (Volvariella volvacea) strain, which has exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-xylanase activities. After single factor experiments, including enzyme concentration, substrate concentration and hydrolysis time, the enzymatic hydrolysis kinetic model was established by response surface analysis. The evaluation indicators of the model involved reducing sugars concentration (RSC), total soluble sugar concentration (TSC), average degree of polymerization (DP) and hydrolysis rate (HR%). The optimized design for continuous enzymatic hydrolysis reactor can be made from the model equation, and the yield of XOs can be predicted. Based on the kinetic model, the hydrolysis behavior of RMFXase was studied. The high-performance liquid chromatography (HPLC) method was used to analyze the end-products produced by RMFXase with oats pelts xylan, bagasse hemicellulose and bagasse separately. The results show that, for the continuous enzymatic hydrolysis reactor, the optimized conditions were enzyme concentration 900 U/mL, substrate concentration 110 mg/mL and reaction time 80 min. Under these conditions，after the bagasse hemicellulose which contained 54.7% xylan were hydrolyzed by RMFCase, the DP value of xylo-oligosaccharides reached to 3 and the substrate hydrolysis rate was 23.94%. The major products in the RMFXase hydrolysates were xylobiose and xylotriose, and the content of xylobiose was higher than that of xylotriose. The yield of xylobiose and xylotriose increased over time, but the yield of xylotetraose initially increased, and then decreased over time, which means that it was degraded to xylobiose. The ?nal degree of polymerization was greater than two. Xylose was not found during the whole reaction period. All of the points above illustrate that RMFXase has a high af?nity for long chain xylan, and that the activity of the RMFXase decreased with the degradation of the long-chain polymers. The results further con?rm that RMFXase has strong endo-β-xylanase activity and no xylosidase activity. The paper also found that the purity of the substrates is another key factor that determines the processing parameters of Xylo-oligosaccharide production.