Objective: The purpose of this experiment was to study the antioxidant activity and mechanism of the bitter peptide RK7 identified in yak milk hard cheese. Method: The bioinformatics methods were used to calculate and analyze the physicochemical properties and biological properties of RK7. The solid-phase synthesis technology was used to synthesize RK7 in vitro and detect its antioxidant activity. The molecular docking tool was used to study the antioxidant mechanism of RK7 based on the signal pathway mediated by longevity enzyme family sirtuins (SIRT1-7) and adenosine monophosphate-activated protein kinase (AMPK). The results showed that the instability index of RK7 was 19.84， the hydrophobicity was 42.86%， the biological activity score was 0.404， human intestinal aabsorption abilty (HIA) was 0.9072+， blood-brain barrier penetration abilty (BBB) was 0.9701-， acute oral toxicity was 0.6259， and the synthetic purity was 99.579%. When the peptide mass concentration changes in the range of 0.2-1.0 mg/mL， the scavenging rate of DPPH free radical was 12.26%-53.78%， and the scavenging rate of ABTS free radical was 80.86%-87.86%. RK7 formed 6 and 5 hydrogen bonds with NAD+ degrading enzymes (3DZK) and cytoplasmic linker protein (2FLU) respectively. The amino acid residues Arg140 and Asp147 of 3DZK and the amino acid residues Ser390 and Asp394 of 2FLU were important active sites for binding to RK7. The combination of RK7 and GSSH with 3DZK and 2FLU showed similar molecular mechanisms， binding to the Site 1 area of 3DZK(X: -11.93， Y: -1.30， Z: 0.38， R: 10 ?魡) and the Site 2 area of 2FLU (X: 8.33， Y: 13.99， Z: 20.41， R: 9 ?魡)， respectively. The above research provided a scientific basis for analyzing and explaining the antioxidant activity of RK7 at the molecular level.