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The effect of hydrothermal conditions on enzymatic hydrolysis lignin (EHL) degradation in water-isopropyl alcohol co-solvent and optimal conditions were investigated. The yields and reactivity toward formaldehyde of degraded enzymatic hydrolysis lignin (DEL) were determined. The optimal conditions of temperature, time, and ratio of solids to liquids were 250 °C, 60 min, and 1:10 (w/v), respectively. The EHL and DEL were characterized by gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR), 1H nuclear magnetic resonance (1H NMR), thermal gravity (TG), and differential scanning calorimetry (DSC) analyses. The results revealed that the molecular weight and polydispersity of DEL were lower than that of EHL. Although the fundamental structure of lignin before and after hydrothermal degradation was retained, the ether (β-O-4, α-O-4, etc.) content decreased, while that of hydroxyl (phenolic and aliphatic) increased. The DTGmax and Tg values shifted from 334 and 117 °C to 304 and 105 °C, respectively.