Background: Sophora tonkinensis Gagnep is an important medicinal plant in China. Previous research has focused on the rapid propagation and quality analysis of in vitro tissue culture plantlets. Few studies have focused on floral bud differentiation. Physiological and molecular mechanisms regulating floral bud differentiation have not been elucidated. Objective: We used paraffin sections and RNA-seq and then analyzed changes in physiological, biochemical, and endogenous hormones to examine floral bud differentiation. Materials and Methods: Buds were harvested in mid-March or early April. After sorting and cleaning, flower buds physiological and biochemical properties were measured and hormones were determined. Morphological observation of S. tonkinensis was carried out using paraffin sections. Results: The transition from the meristematic to the reproductive phase included changes in the shoot apical meristem morphogenesis and cell differentiation. The process includes floral buds initial differentiation and flower primordium differentiation stages; we defined eight stages in total. There was no significant change in soluble content, but soluble protein content gradually decreased. Activities of antioxidant enzymes changed significantly. Total chlorophyll (Chl a + b) content increased significantly. Endogenous hormone levels changed differently during floral bud differentiation. We identified 104,519 expressed genes and 24 were involved in flowering. MADS-box and AP family genes are involved in flower formation and 40 differentially expressed genes associated with floral bud differentiation were identified. Conclusion: The higher soluble sugar, protein, and chlorophyll content and the higher peroxidase activity were beneficial to floral bud differentiation of S. tonkinensis. The dynamic changes in hormone content contribute to differentiating floral buds.