Luteolin, a plant-derived flavonoid, was found to exert effective inhibitory effect to peroxidase activity in a non-competitive manner with an IC 50 of (6.62 ± 0.45) × 10−5 mol L−1. The interaction between luteolin and peroxidase induced the formation of a static complex with a binding constant (K sv) of 7.31 × 103 L mol−1 s−1 driven by hydrogen bond and hydrophobic interaction. Further, the molecular interaction between luteolin and peroxidase resulted in intrinsic fluorescence quenching, structural and conformational alternations which were determined by multispectroscopic techniques combined with computational molecular docking. Molecular docking results revealed that luteolin bound to peroxidase and interacted with relevant amino acid residues in the hydrophobic pocket. These results will provide information for screening additional peroxidase inhibitors and provide evidence of luteolin's potential application in preservation and processing of fruit and vegetables and clinical disease remedy. • HRP activity was inhibited by luteolin in a non-competitive manner. • The thermosensitivity of HRP decreased after treating with luteolin. • The conformation of HRP changed after treating with luteolin. • The surface of structure sharply decreased with treatment of luteolin. [ABSTRACT FROM AUTHOR]