The aim of this study was to investigate the mechanism of interaction between cinnamaldehyde and zein by fluorescence spectroscopy, UV spectroscopy, circular dichroism spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy, so as to provide research basis for improving the mechanical properties, antibacterial properties, and antioxidant properties of zein films. Through three-dimensional fluorescence spectroscopy, it was found that cinnamaldehyde has obvious fluorescence quenching effect on zein, and solvent ethanol has an effect on quenching. When UV-spectroscopy was used to explore the mechanism, it was observed that the absorption intensity of zein in the UV region at 278 nm increased with the increasing concentration of cinnamaldehyde, but the increasing range was not proportional, and the characteristic absorption peak positions of the amino acid residues was not changed. Before and after addition of cinnamaldehyde, the two curves exhibited by circular dichroism were nearly coincident. Using the attenuated total reflection ATR appendage for Fourier transform infrared spectroscopy, we found that after the addition of cinnamaldehyde, the absorption peak at 1 650 cm-1 indicates CO stretching vibration, and 1 538 cm-1 indicates the NH bending vibration in plane. The peak positions at these two sites did not change significantly, but there were obvious acromion peaks at 1 625 cm-1, reflecting the absorption of cinnamaldehyde carbon-carbon double bonds. The peak at 879.44 cm-1 of the fingerprint area disappeared, and a new peak appeared at 973.46 cm-1, reflecting the absorption of the cinnamaldehyde trans double bond, indicating that a non-bonding of zein with cinnamaldehyde. The self-deconvolution calculation of the amide Ⅰ band revealed that the α-helix structure of the zein secondary structure changed little and the β-turn changed significantly. By means of NMR, the chemical shift of proton H in 4 positions changed only 0.01, and after 1 hour and 4 hours of cinnamaldehyde addition, the change of chemical displacement was equal, indicating that the binding reaction of cinnamaldehyde to zein occurred on the surface of the protein and did not cause changes in the secondary structure of zein. The thermodynamic parameters of the system were calculated. It was found that the spontaneous binding reaction occurred between cinnamaldehyde and zein, and the binding ratio of them was 1∶1. When the concentration of cinnamaldehyde was low, the quenching constant decreases with the increase of temperature, but the change was not significant; when the binding constant was very large, the order of magnitude reached 105, and decrease with the increase of temperature. The determination of the fluorescence lifetime of zein in the presence or absence of cinnamaldehyde further confirmed that static quenching occurred between cinnamaldehyde and zein. Comprehensive analysis of various spectra showed that cinnamaldehyde and zein was mainly π—π stacked on the outside of the aromatic region, and it was a combination of electrostatic forces, was static quenching mechanism, and was independent of the action time. The results showed that the combination of cinnamaldehyde and zein do not significantly affect the secondary structure of zein.