Abstract In this study, the effect of traditional acidification and hydrothermal acid hydrolysis on the separation of lignin and xylose from the pre-hydrolysis liquor (PHL) of kraft pulping process was comparatively evaluated. The PHL was acidified at room temperature or hydrothermally treated under acidic conditions to isolate lignin and xylose. As compared with the lignin removal rate of 26.8–32.7% by traditional acidification method, a much higher lignin removal rate of 57.1% was achieved by hydrothermal acid hydrolysis. Meanwhile, the content of xylose in the PHL increased dramatically from 7.91 to 13.91–34.82 g/L after the hydrothermal acid hydrolysis. The hydrophilicity of the obtained lignin gradually increased as evidenced by the decrease of the corresponding water contact angel. Additionally, the thermal stability of the lignin sample increased steadily with the increment of acid dosage. The hydrothermal acid hydrolysis provides an effective approach to fractionate lignin and xylose from PHL.