α-amylase and α-glucosidase are key enzymes in starch digestion and treatment of type Ⅱ glycosuria. The inhibitory activity and interaction mechanism of gardenia yellow（GY） on α-amylase and α-glucosidase were studied by enzyme inhibition kinetics and fluorescence spectroscopy. The results showed that gardenia yellow inhibited α-amylase and α-glucosidase in a competitive manner， and its association constant Kic was 1.47 mg/mL and 0.58 mg/mL， respectively. The fluorescence spectra showed that gardenia yellow had strong quenching ability to the endogenous fluorescence of α-amylase and α-glucosidase， and the mixed quenching of gardenia yellow on α-amylase and α-glucosidase was mainly static quenching through Stern-volmer equation. The changes of enthalpy and entropy showed that the binding of gardenia yellow to glucosidase was mainly driven by Van Der Waals force and hydrophobic interaction， and the binding distance was 4.77 nm and 5.19 nm. Synchronous fluorescence showed that the binding of gardenia yellow with α-amylase and α-glucosidase caused enzyme rearrangement and conformational change， resulting in the change of tyrosine residue or/and tryptophan residue.