Abstract: In order to solve the shortage of xylanase substrate and screen the xylanase which could be used for the enzymatic preparation of xylo-oligosaccharides, firstly, the xylanase activity was detected by replacing the substrate specified in GB/T 23874-2009 "Determination of xylanase activity in feed additives" with xylanase derived from bagasse and beech. At the same time, The xylanase of trichoderma and pichia pastoris was used to hydrolyze different xylan substrates and corncob xylan by enzyme. The components of the enzymatic hydrolysates were analyzed by HPLC, and the enzymatic hydrolysis conditions of corncob xylan were preliminarily optimized. The results showed that compared with the detection results of the substrate specified in GB/T 23874-2009 "Determination of xylanase activity in feed additives", the detection results of beech xylan (Megazyme) were basically consistent with that of the original substrate. The results of bagasse xylan were higher, but the stability was good. Both of them could replace the original substrate for the detection of xylanase activity. By the analysis of HPLC, compared with trichoderma xylanase, the content of xylobiose was higher in the hydrolysis of different substrates by pichia pastoris xylanase, while the content of xylose was opposite. Three kinds of xylanase were used to hydrolyze corncob xylan simultaneously. More than 90% of the products of corncob xylan hydrolyzed by pichia pastoris xylanase were xylo-oligosaccharide, 72.54% and 17.86% of which was respectively xylobiose and xylotriose. being close to the proportion of xylo-oligosaccharide in the products hydrolyzed by Shearzyme 500L xylanase and higher than that by trichoderma xylanase, indicating that it had the application potential in the preparation of xylo-oligosaccharide. The optimal conditions for the hydrolysis of corncob xylan by pichia pastoris xylanase were as follows:reaction temperature of 60℃, pH of 4.8, the concentration of corncob substrate of 0.10 g·mL^-1 and hydrolysis time of 24 h. Under these conditions, the hydrolysis rate of corncob xylan could reach 76%.