The time course of xylo-oligosaccharides concentration and xylo-oligosaccharides yield in the separation of xylo-oligosaccharides from enzymatic hydrolytes was studied using a membrane reactor with constant permeate flux of 4 L · m⁻² · h⁻¹. The results show that xylanases retain 90% of its activity in the reactor. The concentration of xylo-oligosaccharides achieves the maximum, about 5.48 g/L in 30 min. The difference of xylo-oligosaccharides in the retentate and permeate stream is low, <0.62 g/L, therefore it can permeate through membrane. Under the operating conditions that xylan concentration is 30.0 g/L, pH 5.0, operating pressure 16 kPa, temperature 48 °C, feed velocity 400 mL/min, reaction volume 400 mL, enzyme dosage 10% (volume fraction), dilution rate 1 h⁻¹, and enzymatic hydrolysis time 195 min, the yield of xylo-oligosaccharides reaches 31.69%.