Abstract: The xylanase production, selective enzymatic hydrolysis xylan and isolation xylo oligosaccharides by polyacrylamide gel column were studied.The higher activity of xylanase,up to 25.5?IU/mL, was synthesized by T.reesei Rut C30 with improved mandels cultivation broth, and the time of xylanase production was 3 days.When enzymatic hydrolysis lasted for 4～10 hours at 35?g/L substrate concentration, 50?℃ ,pH?4.8, and 1% enzyme dosage,the yield of oligosaccharides and the ratio of oligosaccharides to xylose were 74.08%～87.89% and 16.76～12.30. Xylo oligosaccharides was well isolated by 55?cm × 7.5?cm polyacrylamide gel column.The separating conditions were:injection volume,60?mL,column temperature,50?℃,elution flow rate, 800?mL/h. In this conditions, the whole separating time was only 3 hours.