Abstract: α-Glucosidase is a crucial target for the treatment of type 2 diabetes, and the search for bioactive of α-glucosidase inhibitors holds significant importance. Hesperidin methyl chalcone (HMC) boasts rich bioavailability, metabolic stability, and favorable tissue absorption properties. In the present investigation, a comprehensive suite of experimental and computational techniques were employed to elucidate the inhibitory impact of HMC on α-glucosidase activity as well as to delineate the underlying molecular interaction mechanism. Specifically, enzyme inhibition kinetics were scrutinized to quantify the potency of HMC-mediated α-glucosidase inhibition. Furthermore, fluorescence quenching experiments were conducted to probe the binding interactions between HMC and the target enzyme. Complementary thermodynamic studies were also performed to delineate the driving forces governing the HMC-α-glucosidase association. Finally, in silico molecular docking simulations were leveraged to gain atomic-level insights into the binding mode and key intermolecular interactions dictating the HMC-enzyme complex formation. The IC₅₀ values of HMC and acarbose were (1.39±0.03) mg/mL and (1.84±0.02) mg/mL, respectively, indicating that HMC exhibits superior inhibitory effects on α-glucosidase compared with acarbose. Hydrophobic forces dominated the binding between HMC and α-glucosidase, with a binding site of 1. Synchronous and 3D fluorescence spectra indicated that the interaction between HMC and α-glucosidase resulted in changes in the microenvironment around the tryptophan and tyrosine residues of α-glucosidase, which affected the conformation of the polypeptide chain of the enzyme. The molecular docking results showed that HMC interacted with the amino acid residues of α-glucosidase Glu771, Arg387, Arg428, Asp392, Asp568, Trp391, and Phe384 by the hydrogen bond interaction. In addition, HMC exhibited hydrophobic interactions with numerous hydrophobic residues of the enzymes, including Trp789, Trp715, Trp391, Phe389, Phe385, Arg428, Phe444, Val440, Glu429, Pro441, Lys439, Phe384, Arg387, Asp568, Tyr709, and Glu771. The stronger the binding between α-glucosidase and HMC, the less it binds to the substrates, thereby achieving the purpose of reducing postprandial hyperglycemia.