The xylo-oligosaccharide fluid was purified by chromatographic separation technology with strong acid cation exchange resin as adsorption medium. The optimal separation media and the best separation conditions were determined by comparing different adsorption media and the effects of different factors on the separation of xylo-oligosaccharide. The results showed that xylo-oligosaccharide was well separated by the strong acid cation exchange resin; the optimized separation conditions was as following: flow rate 1.5 mL/min, feed rate 10 mL, feed concentration 30 g/100 mL, operating temperature 70 ℃, the purity was 95.68%, xylo-oligosaccharide was well separated with monosaccharide, which laid a theoretical foundation for the further industrial separation of xylooligosaccharide.