Abstract: In order to optimize a series of process parameters for preparation of xylooligosaccharides from tobacco straw, xylan was extracted by alkaline hydrolysis and xylo-oligosaccharides were prepared by enzymatic hydrolysis. The effects of common factors on the yield of xylan and the yield of xylooligosaccharides were investigated by single factor experiments. For extraction of xylan, 2.000 g straw powder (≤ 100 meshes) was immersed in 20.00 mL solution with a concentration of 24% (m/V NaOH and 1% (m/V) NaBH₄ lye at 70℃ for 4 h, the filtrate was added with 3 times the volume of ethanol for alcohol precipitation and 0.2 times volume of acetic acid to neutralize. For preparation of xylo-oligosaccharides, the optimal conditions were as follows:the pH value of the solution was 5.50, the temperature was 40℃, the time was 6 h, the amount of xylan solution (20 mg/mL) was 10 mL, the amount of xylanase solution was 20 mL (m/V, 4.1 U/mL). Separation and purification of xylo-oligosaccharides were carried out on a cation resin column packed with a height of 18.0 cm and a diameter of 4.5 cm. The purified solution was qualitatively and quantitatively analyzed by high performance liquid chromatography. The results showed that xylo-oligosaccharides with high purity could be obtained by alkali hydrolysis, enzymatic hydrolysis and column filtration. The optimized method is suitable for the preparation and purification of xylo-oligosaccharides from tobacco straw, and has certain reference value for optimizing the process of preparing xylo-oligosaccharides in industry.