AbstractBall-milled bamboo (Neosinocalamus affinis) was first treated under ultrasound at 20 oC in 95% ethanol solution for 0 to 50 min, dissolved in sodium hydroxide/urea solution (7% NaOH/12% urea) at –12 oC, and then extracted with ethanol and dioxane to isolate lignin. The structure of the isolated lignin was characterized with a set of wet chemical and spectroscopic methods, including UV, FT-IR, 13C NMR, and HSQC spectroscopies. The results showed that the lignin extracted from bamboo consisted of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) type lignins with minor cinnamate units. The predominate lignin inter-units were β-O-4´ ether linkages, followed by phenylcoumaran and a lower proportion of resinol and spirodienone. It was also found that the ester groups of lignin were cleaved during the pretreatment process with cold alkaline solution.