The protection of Cumalic acid (CA), antioxidant, in the biochemical process in nature has aroused great interest.Polyphenol oxidase (PPO), an enzyme, plays a vital function in aging and browning of plants, such as vegetables, fruits and mushrooms. The interaction of CA and PPO reveals the important information in metabolism and aging. Thus, the molecular mechanism of CA binding with polyphenol oxidase (PPO) was explored by combining spectroscopic methods with molecular modeling. A three-dimensional fingerprint of the CA-PPO complex was built for the first time to characterize the interaction biopolymer between CA and PPO. Application of the spectroscopic methods indicated that CA effectively quenched the intrinsic fluorescence of PPO. The enthalpy change (ΔH°) and entropy change (ΔS°) suggested that the CA-PPO complex was predominantly stabilized by hydrophobic interactions CA and PPO. Building the λ-UV-F fingerprint of CA-PPO made it possible to demonstrate the three-dimensional interactions between CA and PPO. Subsequently, molecular modeling demonstrated that CA was primarily bound to PPO by hydrophobic interactions and hydrogen bonds located at amino acid residues Ala202, His38, His54 and Ser206. The computational simulations were consistent with the spectral experiments demonstrating confidence in the three-dimensional model determined of the CA-PPO interaction.