α-Glucosidase is related to the increase in postprandial blood glucose in vivo. Inhibition of α-glucosidase is supposed to be an effective approach to treat type 2 diabetes mellitus (T2DM). Trilobatin, a member of the dihydrochalcone family, shows anti-oxidant, anti-inflammatory and anti-diabetic activities. In this study, the inhibitory activity and mechanism of trilobatin on α-glucosidase were investigated using multispectroscopic and molecular docking techniques. The kinetic analysis showed that trilobatin reversibly inhibited α-glucosidase in a noncompetitive-type manner and the value of IC50 was 0.24 ± 0.02 mM. The analysis of fluorescence spectra demonstrated that the formation of the trilobatin-α-glucosidase complex was driven mainly by hydrogen bonding and van der Waals forces, resulting in the conformational changes of α-glucosidase. Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD) measurements suggested that the interaction could change the micro-environment and conformation of α-glucosidase affected by trilobatin. Molecular docking analysis determined the exact binding sites of trilobatin on α-glucosidase. These results indicated that trilobatin is a strong α-glucosidase inhibitor, thus it could be conducive to ameliorate T2DM.