Abstract: This study aimed to screen and identify peptide sequences with potent antioxidant properties from hemp seeds and to evaluate their gastrointestinal stability and intestinal absorption permeability. Hemp seed protein was used as the raw material to prepare hemp seed peptides (HSPs). Peptides exhibiting high antioxidant activity were screened and isolated using a combination of ultrafiltration, Sephadex G-15 gel filtration, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro simulated gastrointestinal digestion and Caco-2 cell monolayer transport assays were conducted. The results demonstrated that the F1 fraction exhibited the strongest antioxidant activity following ultrafiltration and Sephadex G-15 separation. Three HSPs sequences were identified through LC-MS/MS coupled with virtual screening. Among them, the in vitro simulated gastrointestinal digestion and Caco-2 cell transport assays demonstrated that the peptide Phe-Tyr (FY) displayed good gastrointestinal stability, with an apparent permeability coefficient (P_app) of (6.017±0.4998)×10⁻⁷ cm/s, classifying it as a moderately absorbed substance. This study proposes a novel strategy for the high-value utilization of hemp seed protein and offers scientific evidence supporting the potential in vivo efficacy of specific hemp seed peptides upon oral administration.