Abstract: In the preliminary experiment, a water-soluble peptide fraction was extracted from self-developed Yunnan Binglangjiang buffalo milk cheese, which exhibited good in vitro antioxidant activity through in vitro activity evaluation. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology was further employed to identify and screen novel antioxidant peptides, to elucidate their structural characteristics, and to reveal their potential interaction mechanisms with target proteins through molecular simulation approaches. The results showed that a total of 1,614 peptide sequences were identified from the water-soluble peptide fraction with molecular weight less than 3 kDa by mass spectrometry, which were mainly derived from αs1-casein, αs2-casein, β-casein, and κ-casein. Among them, peptides with molecular weight less than 1,500 Da accounted for the majority (63.14%). Based on the results of bioactive peptide database search, relative peptide abundance, bioactivity prediction, and molecular docking binding energy analysis, three peptides with the sequences WPQYLK, PFPGPIPK, and FPGPIPK were screened out. Their IC₅₀ values for DPPH· scavenging were 2.61, 3.64, and 2.66 mg/mL, respectively. Fourier transform infrared spectroscopy analysis revealed that the secondary structure of WPQYLK was mainly composed of β-turn, random coil, and β-sheet, while PFPGPIPK and FPGPIPK were mainly composed of β-turn and β-sheet. Molecular docking and molecular dynamics simulation analyses indicated that all three antioxidant peptides could bind tightly to Keap1 through hydrogen bonds and hydrophobic interactions, among which WPQYLK formed a more stable complex with Keap1. These findings may provide a reference for the development of high value-added buffalo milk cheese and dairy-derived antioxidant peptides.