A method for the isolation of lignin in high purity from cotton stalk is presented in this study. Lignin fractions were obtained by dissolution of ball-milled cotton stalk in lithium chloride/dimethylacetamide (LiCl/DMAc), tetrabutylammonium fluoride/dimethylsulfoxide (TBAF/DMSO), lithium chloride/N-methyl-2-pyrrolidone (LiCl/NMP), NaOH/urea/H2O, NaOH/thiourea/H2O, and 1-allyl-3-methylimidazolium chloride/dimethylsulfoxide ([Amim]Cl/DMSO) followed by precipitation in the course of which lignin and carbohydrate fractions were separated. Structural elucidation of the lignin fractions was investigated by sugar analysis, FT-IR, and 1D/2D NMR spectroscopy. The results showed that the separated lignin fractions possessed higher purities and molecular weights than MWL. 1D/2D NMR spectra demonstrated that guaiacyl (G) and syringyl (S) units were predominant in the lignin fractions, and a small amount of p-hydroxyphenyl (H) unit was also detected. Moreover, the separated lignin fractions consisted mainly of beta-O-4' aryl ether linkages (0.36-0.61/Ar), followed by resinol (0.05-0.08/Ar) and phenylcoumaran structures (0.03-0.05/Ar). In consideration of the relatively high yield and purity and limited structural changes, isolation of lignin from ball-milled materials by complete dissolution in [Amim]Cl/DMSO provided us a more effective approach as compared with the other systems.