Bamboo is a swiftly renewable natural material. A thorough exploration of its functional hierarchical structure is helpful to improve its utilization efficiency. Previous studies have found that there are notable structural distinctions in bamboo fibers and parenchyma cell wall at the nanoscale. The fluctuation in cellulose microfibrils (CMF) within bamboo has been verified as a critical factor significantly impacting the mechanical properties of bamboo fibers. This study systematically examines the radial gradient variation in the cellulose supramolecular structures of bamboo culms through synchrotron X-ray scattering. The measured cellulose crystallinity index (CrI), the diameter and packing distance of CMF, the orientation parameter, and the distribution of microfibril angle (MFA) were found to be correlated with the radial distribution of fibers and parenchyma cells. In the radial direction, the relative CrI values of 004 reflection increased from 26 % for the innermost layer to 51 % for the outermost layer; the CMF diameter decreased from 2.75 nm to 2.66 nm; the packing distance of CMF’ cylinders was reduced from 3.68 nm to 3.35 nm. The measured mean MFA values exhibit a decrease from approximately 70° to around 5°. Moreover, the azimuthal X-ray scattering signals were effectively segregated from fiber and parenchyma cell wall using the non-negative matrix factorisation (NNMF) method. This methodology holds promise for nanostructure analysis in complex plant stems comprising fiber and parenchyma cells, including but not limited to wheat, corn stalks, and other biomaterials.