Casein (CN) has been regarded as an excellent protein source for preparing bioactive peptides. In this study, the casein peptides released in the mouse gastrointestinal tract were evaluated. The 10-week-old mouse was orally administered with 5 mg casein. After 0.5 h, the peptides in the stomach and small intestine of the mouse were extracted and analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). A total of 343 peptides were identified, and 98, 36, 181 and 28 peptides were derived from αs1-, αs2-, β- and κ-CN respectively. Then, in silico methods were adopted to predict the potential anticoagulant peptide, including PeptideRanker, Innovagen. A novel anticoagulant peptide, AVPYPQR (β-CN, fragment 177-183), was screened and its anticoagulant activity was verified. In vitro anticoagulant assay showed that the peptide AVPYPQR can observably prolong activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), which indicated that the peptide AVPYPQR exerts its anticoagulant activity in the intrinsic, extrinsic, and common pathways. Meanwhile, the cell viability of this peptide was estimated on the human umbilical vein endothelial cells (HUVECs). The physicochemical characteristics of this peptide have been assayed by PepDraw and ExPASy-ProtParam. The study indicated that casein could be a valuable source for preparing bioactive peptides by gastrointestinal (GI) tract digestion.