Abstract: Xylobiose was prepared by xylanase hydrolysis of birchwood xylan in the PEG6000/sodium citrate aqueous two-phase system (ATPS). The optimized conditions for enzymatic hydrolysis in ATPS were birchwood xylan 10 g/L, xylanase 20 IU/g (calculated by the mass of xylan), 50 ℃ and pH 5.6. The enzymatic hydrolysis yield of xylobiose was 13.8% for 8h. The recoveries of xylanase and xylobiose were 66.4% and 87.2%, respectively. Then, purification of xylobiose was carried out on Sephadex LH-20 with using degassed high purified water as eluent. The optimized conditions for purification of xylobiose were column temperature 35 ℃, 0.1 mL/min and sample volume 0.3 mL. After purification, the purity of xylobiose was improved to 98.5% under the optimized conditions.