Bamboo is a natural fiber composite with layered structure. Millions of years of evolution have endowed bamboo with the most effective structure in nature. The ingenious microstructure provides bamboo with excellent mechanical properties. Bamboo culm is composed of the cortex, a middle layer, and a pith ring. The cortex refers to the area starting from the periphery of the culm wall to the vascular bundles. The present study obtained the two-dimensional microstructure of bamboo cortex cells by optical microscopy and characterized the three-dimensional structure through high-resolution X-ray microtomography (µCT). Based on the analysis, the bamboo cortex cells were classified into four layers: epidermis layer, hypodermis layer, transitional layer, and parenchyma layer. The average pore volume of the bamboo cortex was about 1.54×10-6 mm3, the porosity was 36.1%, and the relative density was 0.639. The epidermis layer, hypodermis layer, transition layer, and parenchyma layer cells had a cell cavity volume of 917.81 µm3, 714.22 µm3, 1258.19 µm3, and 3117.65 µm3, respectively, an average length3d (L) of 19.38 µm, 25.84 µm, 26.46 µm, and 34.88 µm, respectively, an average breadth3d (W) of 14.11 µm, 9.44 µm, 15.22 µm, and 16.6 µm, respectively, and sphericity of 0.85, 0.76, 0.75, and 0.78, respectively. Studies on bamboo anatomical structure, especially three-dimensional digital characterization, will enrich the bamboo microstructure database. Besides, the three-dimensional structure of the bamboo cortex revealed in this study can provide a reference for optimizing composite material hierarchy and biomimetic design.