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The separation characteristics of Eucalyptus camaldulensis biomass cell wall were investigated and analyzed by FTIR, UV and XRD. The result showed that the crystallinity of untreated sample were lower than ones of treated samples at 10° C, 20° C and 30° C. Effect of temperature was very notable so as to use the lower temperature if cellulose would be kept natural structure during lignin separation from lignincellulose. Treatment time and temperature had a notably significant effect on SLR of E. camaldulensis lignin cellulose, and the SLR increased gradually with the temperature increment, and SLR were 73.35% at 10° C, 80.14% at 20° C, 83.73% at 30° C. The maximum of SLR increased with the rising of temperature 10° C, 20° C and 30° C. Consequently, the best separation conditions of lignin were 24h and 30° C. During hemicelluloses separation, the peak of C=O disappeared, and the peak of CO-OR, O-H and C-O abated, resulting that acetyl in hemicelluloses were mainly broken by the dehydration, heat decomposition reaction and mild thermal degradation. During lignin separation, the side chain and benzene ring of lignin of E. urophynis lignin cellulose reached the largest bond breaking characteristics under the temperature of 10° C at 7h, 20° C at 7h and 30° C at 24h.