A mild and successive method was proposed and evaluated for fractionating lignin samples from Eucalyptus globulus. The extraction efficiency and structural features of the extracted lignin samples were comprehensively investigated by a yields analysis, 1H-nuclear magnetic resonance (NMR), 13C-NMR, and two-dimensional heteronuclear single quantum coherence (2D-HSQC) NMR. The catalyzed ethanol systems consisting of ethanol, 4-methyl-2-pentanone, dimethyl sulfoxide (DMSO), formic acid, and distilled water were effective for extracting lignin from Eucalyptus globulus, and the yield of lignin was 75.2% when the extraction process was held at 145 °C for 180 min. Compared with that of the milled wood lignin (MWL), the NMR spectra of the extracted lignin fractions supported the destruction of β-O-4 units for the disappearance of Hα signals. Moreover, the striking characteristics of the extracted lignin were the destruction of ether bonds of S3,5 and the condensation of syringyl (S) and guaiacyl (G) units. In short, lignin fractions with relatively complete structures were effectively extracted with the catalyzed ethanol systems.