This study focuses on the effective separation of cellulose from bamboo through a two-step process.Several organic alkalies 2-pyrrolidinone, ethylurea, dibutylamine, N-methylformamide and tetramethyl guanidine were used to remove lignin and isolate cellulose from massive bamboo. The results showed that tetramethyl guanidine can effectively remove lignin and hemicellulose while retaining almost all the cellulose in the residual solid. The removal of lignin and hemicellulose achieved 86.0% and 84.0% after heating for 3h at 150°C, respectively. Subsequently, the nal remove of lignin and hemicellulose increased to 91.5% and 93.8%, respectively, after a simple alkaline H 2 O 2 bleach treatment. Interestingly, the loss of cellulose was very small after two-step treatments, and 96.9% of the component was still retained. The crystalline increased from 61.3-68.4% in XRD graphs due to the remove of lignin and hemicellulose. The SEM images indicated that the diameter of cellulose bundles decreased from 80-100µm to about 50µm after organic alkali treatment, and then the ber bundle was completely separated into a single long ber with a diameter of about 10 µm after H 2 O 2 bleaching. The FT-IR spectra con rmed the high selective removal of lignin and hemicellulose. Two-dimensional 1 H-13 C NMR (2D HSQC) spectra were analyzed to investigate the lignin structure, and found that only the signals of -OCH 3 , C γ -H γ in β-O-4' (A γ ) and β-β' (C γ ) structures and C 5 -H 5 in guaiacyl (G 5 ) did not disappear after two-step treatment.