Abstract: To investigate the presence of bioactive peptides in milk pimples, Xinjiang milk pimples were utilized as raw materials for the preparation of peptides with varying molecular weights via ultrafiltration separation. Peptidomics, in conjunction with bioinformatics, was employed to identify and screen the milk pimple peptides, followed by in vitro activity studies. Additionally, the stability of these peptides was examined, and their mechanisms of action were elucidated through molecular docking. The results indicated that the ultrafiltration peptide with a molecular weight of less than 3 kDa exhibits significant ABTS⁺ free radical scavenging activity (IC₅₀=1.79 mg/mL) and α-glucosidase inhibitory activity (IC₅₀=3.48 mg/mL). Utilizing LC-MS/MS, a total of 376 peptides, primarily derived from β-casein and α_S1-casein, were identified. Through a combination of bioinformatics analysis and in vitro activity experiments, an antioxidant peptide, FPKYP (IC₅₀=23.55 μg/mL), and an α-glucosidase inhibitory peptide, EMPF (IC₅₀=3.24 mg/mL), were successfully screened. The results of the stability study indicate that despite exposure to varying temperatures, pH levels, and gastrointestinal digestion treatments, the activity of peptide FPKYP was preserved at approximately 50%. In contrast, the activity of peptide EMPF significantly increased to over 80% following gastrointestinal digestion. Molecular docking results indicated that FPKYP and EMPF primarily interact with the active amino acid residue sites of Keap1 (Val369, Val465, Val467, and Val418) and α-glucosidase (Val335, Val334, Pro230, and Gly228), respectively, through hydrogen bonds and hydrophobic interactions. These compounds tightly bind to exert antioxidant activity and inhibit α-glucosidase activity. Milk pimple peptide had good antioxidant and α-glucosidase inhibitory activities, which provides a basis for further development of milk pimple functional food.