Recalcitrance of lignocellulosic biomass is closely related to the presence of lignin in secondary cell walls, which has a negative effect on enzyme digestibility, biomass-to-biofuels conversion, and chemical pulping. The lignification process and structural heterogeneity of the cell wall for various parts of moso bamboo were investigated. There were slight differences among three different column parts of moso bamboo in terms of chemical compositions, including cellulose, hemicelluloses, and lignin. However, the detailed analysis of the fractionated lignin indicated that the acid-soluble lignin was first biosynthesized, and the largest molecular weight value was detected from the bottom part of the moso bamboo, as well as the highest syringyl-to-guaiacyl ratio. Although the main β-O-4 aryl ethers and resinol structures were clearly present in all lignin samples examined by NMR analysis, the relatively small lignin biomacromolecule in the top part of the moso bamboo lead to poor thermal stability. For the bioconversion process, no significant difference was found among all the moso bamboo samples, and the relatively higher hydrolysis efficiency was largely dependent on the low crystallinity of cellulose rather than the degree of lignin biosynthesis.