Inhibition of glycosidase activity represents a promising therapeutic strategy for managing diabetes mellitus. The current study delineated the inhibitory effects and mechanisms of daidzein and genistein on two key glycosidases, α-amylase and α-glucosidase, employing a multifaceted approach encompassing activity assay, enzymatic kinetics, multi-spectroscopy, molecular docking, and in vivo sucrose-loading test assays. Our findings revealed that daidzein and genistein acted as a potent reversible mix-type inhibitors against α-amylase, and reversible non-competitive inhibitors against α-glucosidase. Spectroscopic analyses (UV-vis, fluorescence quenching, synchronous fluorescence, 3D fluorescence, CD, and ANS spectra) indicated daidzein and genistein's binding induced conformation and microenvironment alterations of two enzymes, affecting their activities. Molecular docking substantiated interaction forces between daidzein and genistein and two enzymes. Moreover, an in vivo sucrose-loading test in mice demonstrated genistein's efficacy in reducing postprandial blood glucose levels. Collectively, these insights underscored the potential of daidzein and genistein as a hypoglycemic dietary supplement, offering a natural alternative for diabetes management.