Abstract: To investigate the α-amylase inhibitory activity and molecular mechanisms of major isoflavones from Pueraria lobata, inhibitory kinetics was used to evaluate their inhibitory effects on α-amylase. Fluorescence spectroscopy, synchronous fluorescence, and circular dichroism were utilized to observe the spatial structure and stability changes of α-amylase upon binding to these isoflavones. Molecular docking was further performed to explore the intermolecular interactions. The results revealed that both puerarin (8-C-glycoside substitution, IC₅₀=0.489±0.096 mg/mL) and daidzin (7-O-glycoside substitution, IC₅₀=1.216±0.152 mg/mL) spontaneously bound to the enzyme through mixed-type inhibition, which primarily occurred through hydrogen bonds and van der Waals forces. Their binding significantly enhanced the polarity of the Trp/Tyr residue microenvironment and induced secondary structural rearrangements, leading to a loosened enzyme conformation. The 8-C-glycosyl group of puerarin precisely embedded into the active center of the enzyme, forming a hydrogen bonding network synergized with π-π stacking and hydrophobic interactions to enhance binding stability. In contrast, the 7-O-glycosyl group of daidzin caused steric hindrance, displacing its B-ring from the binding pocket and weakening its interactions. This study elucidated the molecular mechanism of Pueraria lobata isoflavones in inhibiting α-amylase, thereby revealing the structural superiority of 8-glycoside substituted isoflavones in targeting the active pocket of the enzyme. These findings provide novel insights into the hypoglycemic functional activities of Pueraria lobata isoflavones.