Organic acid pretreatments can efficiently separate biomass-based hemicellulose and selectively produce hemicellulose-derived sugars. In this study, hemicellulose is separation as xylose, oligosaccharides in the tropic acid-catalyzed hydrothermal pretreatment of eucalyptus. The maximum yield of hemicellulose-derived sugars (85.78 %) with 71.25 % xylose selectivity (based on the total xylose in raw material) was achieved in the hydrolysate under optimal conditions (5 % TA, 160 ℃, 80 min). The yield of hemicellulose-derived sugar and the separation yield of hemicellulose increased by 11.06 % and 11.45 % compared with glycolic acid pretreatment in the similar severity factor. The separation yield of cellulose and lignin was decreased by 4.23 % and 0.98 %, respectively. This resulted in residual solids with higher biological stability (higher fiber crystallinity index, higher thermal stability, and higher lignin content). Therefore, higher hemicellulose separation selectivity and rich hemicellulose-derived sugars were obtained using TA pretreatment. The work would bring up a new method for biomass refining.