The dried pseudobulbs of Bletilla striata, an important traditional Chinese medicine record as BaiJi, have an extraordinary polysaccharide content and excellent prospects for medicinal effects. However, the distribution and molecular mechanism underlying biosynthesis are poorly understood. In this study, Chemical and immunologic analyses was performed in representative tissues of B. striata, and the results showed that the conventionally called B. striata polysaccharides (BSPs) are water-soluble polysaccharides deposited only in pseudobulbs. The structural component of BSPs is glucomannan, with a mass ratio of approximately mannose:glucose = 3:2. The BSPs are presented in the parenchyma of the pseudobulbs known as glucomannan idioblasts and distributed in the cytoplasm within cellular membranes but not contained in the vacuole. Comparative transcriptomics and bioinformatics analyses mapped the pathway from sucrose to BSP and identified BsGPI, BsmanA, and BsCSLAs as the key genes of BSP biosynthesis, suggesting that the functional differentiation of CSLA may be critical for the flow of glucomannan to the BSP or cell wall. Subsequently, virus-mediated gene silencing showed that silencing of two CSLA (Bs03G11846 and Bs03G11849) led to a decrease in BSP content, and yeast two-hybrid and luciferase complementation experiments confirmed that four CSLAs (Bs03G11846, Bs03G11847, Bs03G11848, and Bs03G11849) can form homo- or heterodimers, suggesting that multiple CSLAs may form a large complex function for BSP synthesis. Our results provide cytological evidence of BSP, isolation and characterization of candidate genes involved in BSP synthesis, laying a solid foundation for further research on its regulation mechanisms and genetic engineering breeding mechanisms of B. striata.