This study aimed to reveal the mechanism of action of oligopeptides KNGP on cyclooxygenase-2 （COX-2）， and to explore the interaction between KNGP from yellow pond turtle peptides （YPTP） and COX-2.The IC₅₀ values of YPTP， synthesized KNGP and RG were determined by UV spectrophotometry.The mechanism of interaction between KNGP and COX-2 was investigated by intrinsic fluorescence， synchronous fluorescence， three-dimensional fluorescence spectroscopic techniques and ANS fluorescence probe.The mode of action between KNGP and COX-2 was studied by molecular docking.The results showed that the IC₅₀ values of YPTP， KNGP and RG were 0.609， 0.046 and 0.056 mg/mL， respectively.The IC₅₀ of KNGP was lower than that of the positive drug ibuprofen （IC₅₀=0.217 mg/mL）， indicating that KNGP had a significant inhibitory effect on COX-2 enzyme.The intrinsic fluorescence results showed that KNGP had a static quenching effect on COX-2， and the action site was single.Meanwhile， the results of thermodynamic parameter calculations showed that hydrophobic interaction is the main driving force in the process of KNGP binding to COX-2， and KNGP binding to COX-2 is a spontaneous process driven by entropy.The results of synchronous fluorescence， three-dimensional fluorescence spectroscopic and ANS fluorescence probe showed that the hydrophobic environment of tyrosine and tryptophan in COX-2 changed， and the hydrophobicity of COX-2 surface was weakened.The molecular docking between KNGP and COX-2 showed that KNGP molecules formed hydrogen bonds and hydrophobic interactions with the amino acid residues of COX-2 active center mainly through NH， C=O structure， indole and imidazole structure， and enhanced the binding stability by forming electrostatic interaction.Therefore， the results showed that KNGP could form a complex with COX-2 at a single site through hydrogen bonding and hydrophobic interaction， and change the secondary structure of the enzyme to inhibit its activity.