High selectivity of hemicellulose separation is difficult to achieve using organic acid pretreatment. This is attributed to the depolymerization and polycondensation of lignin, which is accompanied by the separation of hemicellulose. In this study, the separation of eucalyptus components was studied by 1-amino-2-naphthol-4-sulfonic acid (ns-A) pretreatment. An organic acid concentration of 9% at a pretreatment temperature of 170 °C performed for 80 min was determined as the optimum condition for ns-A pretreatment. The separation yield of hemicellulose increased to 82.76% compared with that of p -toluenesulfonic acid pretreatment. The separation yield of lignin decreased from 30.20% to 8.43%. The surface coverage of lignin increased from 0.70% to 0.83%. Furthermore, the recovery yield of the organic acids in the ns-A pretreatment process was 91%. The ns-A-pretreated acid solution provided stable, efficient separation within six reuse cycles. Lignin in eucalyptus was found to be rich in aliphatic hydroxyl groups and guaiacyl-syringyl units. Therefore, the depolymerization and polycondensation of lignin was inhibited while efficiently separating hemicellulose during ns-A pretreatment. This study provides a new method for the efficient separation of lignocellulosic biomass. [Display omitted] • The main eucalyptus components were separated by1-amino-2-naphthol-4-sulfonic acid pretreatment. • The separation yield of hemicellulose was 82.76% under the optimum conditions. • The lignin separation yield decreased to 8.43% under the optimum conditions. • The depolymerization and polycondensation of lignin was inhibited. [ABSTRACT FROM AUTHOR]