Abstract: This study investigated the gene expression differences between the fruiting body and mycelium stages of Pleurotus ferulae through transcriptome sequencing and bioinformatics analysis, focusing on the molecular mechanisms underlying fruiting body development and polysaccharide biosynthesis. The results revealed that 6458 genes were co-expressed in both fruiting body and mycelium, while the mycelium exhibited a significantly higher number of unique genes (12218) compared to the fruiting body (1022), suggesting that fruiting body-specific genes predominantly drive morphogenesis and the biosynthesis of bioactive compounds. Further comparative analysis using the mycelium transcriptome as a control identified 5339 significantly differentially expressed genes (DEGs) (60 upregulated, 5279 downregulated) in the fruiting body transcriptome. These DEGs were enriched in pathways related to carbohydrate metabolism, energy metabolism (including oxidative phosphorylation and the TCA cycle), and genetic information processing, revealing a substantial activation of metabolic activity during fruiting body development. Furthermore, 9 polysaccharide biosynthesis-related pathways were pinpointed from 73 KEGG pathways, and 23 key enzyme genes were identified. These genes were primarily associated with pyruvate metabolism, starch and sucrose metabolism, glycolysis/gluconeogenesis, and amino sugar/nucleotide sugar metabolism, thereby delineating core metabolic nodes in polysaccharide biosynthesis. This study pioneered the molecular regulatory framework governing the development and polysaccharide biosynthesis in Pleurotus ferulae. It provided critical theoretical foundations and genetic resources for elucidating the synthesis mechanisms of bioactive polysaccharides, enhancing cultivation efficiency and processing adaptability for food applications, and developing molecular marker-driven quality improvement techniques. These findings laid a molecular foundation for functional food development (e.g., polysaccharide-based thickeners and nutritional enhancers) and the targeted breeding of high-value-added mushroom products tailored to the food industry.