<p>Bamboo exhibits fiber lengths comparable to those of softwood species, making it a high-quality raw material for long fibers. However, the effective separation and utilization of these long fibers are severely impeded by the parenchyma tissue present in bamboo. Therefore, the efficient separation of vascular bundles and parenchyma tissue from bamboo is a crucial prerequisite for achieving high-value utilization of its long fibers. This study systematically evaluates and quantifies the impact of alkaline conditions on the selective separation of bamboo vascular bundles (VB) and parenchyma cells (PC) through single-factor experiments, reveals the microscopic mechanisms that induce selective removal of PC, and proposes a mild pretreatment method that balances yield while preserving fiber integrity. The VB and PC samples obtained at different treatment temperatures were thoroughly characterized in terms of microstructure, chemical composition, crystallinity, and thermal stability. The results indicated that alkali treatment removed lignin from the cell corner middle lamella and compound middle lamella of bamboo, facilitating the separation of VB and PC. When PC separation efficiency reached its maximum (10.28%), VB separation efficiency was 65.64%. Under these conditions, the crystallinity index, and cellulose content of the samples were significantly increased. PC was found to have a higher syringyl/guaiacyl (S/G) ratio and a lower content of condensed lignin linkages, indicating greater reactivity under alkaline conditions. This study elucidates the mechanistic differences in alkali-induced separation of two tissue types for the first time, providing empirical data and a theoretical foundation for optimizing the efficient separation process of VB and PC.</p>