Casein hydrolysates exert various biological activities and the responsible functional peptides are being continuously identified from them. In this study, tryptic casein hydrolysate was fractionated by ultra-filtration membrane (3 kDa), and the peptides were identified by capillary electrophoresis time-of-flight tandem MS (CE-TOF-MS/MS). Then, in silico methods were used to analyze the toxicity, solubility, stability, and affinity between the peptides and angiotensin I-converting enzyme (ACE). Finally, a new ACE-inhibitory (ACEI) peptide, EKVNELSK, derived from the α_S1-casein (fragment, 35–42), was screened. The IC₅₀ value of the peptide was 5.998 mM, which was determined by an HPLC method. The Lineweaver-Burk plot indicated that this peptide is a mixed-type inhibitor against ACE. Moreover, Discovery Studio 2017 R2 software was adopted to perform molecular docking to propose the potential mechanisms underlying the ACEI activity of the peptide. These results indicated that EKVNELSK is a new ACEI peptide identified from casein hydrolysate.