The mechanism of interaction between CTC and PEP was investigated by using fluorescence spectra, UV-Vis absorption spectra, circular dichroism (CD), 3D fluorescence spectra, synchronous fluorescence spectra and molecular docking methods.The quenching mechanism associated with the CTC-PEP interaction was determined by performing fluorescence measurements at different temperatures. The binding constants (KA) at three temperatures (298, 303, and 308 K) were 4.345×107, 2.836×107 and 1.734×107 L·mol-1 respectively, and the number of binding sites (n) was 1.618, 1.587, and 1.555, respectively. The n value was close to unity, which meant that there was only one independent class of binding site on pepsin for CTC. Based on the thermodynamic analysis, thermodynamic parameters at 298 K were calculated as follows: ΔH (-70.13 kJ·mol-1), ΔG (-43.57 kJ·mol-1), and ΔS (-89.00 J·(mol·K)-1). It was known from ΔH<0 and ΔS<0 that Van der Waals’ forces and hydrogen bonds were the main forces between CTC and PEP, the reaction was spontaneous from ΔG<0. According to Frster’s dipole-dipole non-radiative energy transfer theory, the specific binding distance of CTC-PEP system was 3.240 nm, it proved that there was non-radiative energy transfer between CTC and PEP. Molecular docking further suggested that CTC molecule bound within the active pocket of PEP. There were the van der Waals forces between CTC and residues VAL30, SER35, TYR189, THR74, THR77, GLY78 and LEU112 of PEP, and hydrogen bonds between CTC and GLU13, GLY217, ASP32, ASP215 and GLY76. There also was a hydrophobic interaction between CTC and the amino acid residue TYR75 of PEP. Various forces make CTC and PEP form a stable complex.The effects of CTC on the conformation of PEP were analyzed by UV absorption spectroscopy, synchronous fluorescence spectroscopyand 3D fluorescence spectroscopy. It is demonstrated in detail that CTC can increase microenvironment polarity and decrease the hydrophobicity of tryptophan (Trp) residues in PEP. Circular dichroism spectra indicated the secondary structure of PEP was partially changed by CTC with the percentage of α-helix increasing from 11.6% to 21.0% andthe percentage of β-sheet decreasing from 47.3% to 28.2%.The content of β-Turnstructure increased from 19.6% to 24.2%, and the content of Random coil increased from 27.6% to 34.2%, indicating that CTC interacted with PEP, and CTC changed the microenvironment around PEP, and also changed the secondary structure of PEP. The results of this study are helpful to understand the binding mechanism of CTC and PEP, and provide an important basis for the rational use of CTC.