Abstract: In order to improve the enzymatic hydrolysis efficiency of poplar xylan to xylo-oligosaccharides and reduce the cost of enzymatic preparation of xylo-oligosaccharides, this study focused on the induced expression conditions of Bacteroides-derived xylanase PdoXyn10A produced by high-density fermentation of recombinant Escherichia coli (PET-PdoXyn10A-DE3) and the process conditions of enzymatic hydrolysis to prepare xylo-oligosaccharides. The effects of the inducer concentration, the induced optical density (OD₆₀₀) value, induction temperature and induction pH value on the production of recombinant xylanase PdoXyn10A by PET-PdoXyn10A-DE3 were studied under 5 L fermenter level. The effects of reaction temperature and pH value on the enzyme activity of corncob xylanase during hydrolysis and the effects of enzyme addition and hydrolysis time on the preparation of xylo-oligosaccharide from poplar acetic acid hydrolysate were determined. The results showed that the activity of recombinase reached 362.67 U/mL and the biomass of recombinant Escherichia coli reached 28.83 g/L under high density fermentation at the optimal induction conditions with 0.25 mmol/L of inducer concentration, induction OD₆₀₀ value of 55, induction temperature 34 ℃ and induction pH value 7.0. The optimum conditions for the preparation of xylooligosaccharides by enzymatic hydrolysis were as follows: enzyme reaction temperature of 40 ℃, pH value of 5.2, the enzyme dosage of 12 U/mL, and reaction time of 6 h. Under these conditions, the content of xylo-oligosaccharides was 3.21 g/L, the content of xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose were 1.09, 0.97, 0.84, 0.18, 0.13 g/L, respectively, and the content of xylose was 1.82 g/L.