In order to optimize the technological conditions for preparing ACE inhibitory peptide of sheep casein by enzymatic hydrolysis， and a new ACE inhibitory peptide was screened and identified from casein hydrolysate. ACE inhibitory peptide was prepared from sheep cheese protein by five proteases enzymatic hydrolysis， and taking molecular weight distribution， degree of hydrolysis and inhibition rate of angiotensin-I-converting enzyme (ACE) as indicators to screening the most suitable protease. The single factor and response tests were used to optimize enzymatic hydrolysis conditions. The amino acid sequences of components less than 3 ku were identified by Orbitrap Fusion Lumos Tribrid MS， and the potential ACE inhibitory peptides were screened and determining their IC50 values after synthesis. The inhibition kinetics of enzyme was determined by Linewaver-Burk mapping， and molecular docking was used to explain the mechanism of peptides inhibition of ACE. The results showed that alkaline protease was the most suitable protease， the optimal enzymolysis conditions were as follows: pH 6， substrate concentration of 8%， enzyme/substrate ratio(E/S) of 4%， temperature at 55 ℃ and enzymatic hydrolysis time for 90 min. The ACE inhibition rate of casein antihypertensive peptide prepared under this condition was 99.1%. A total of 484 peptides were identified in the fraction less than 3 ku， among which 10 peptides had been verified to have ACE inhibitory activity. A novel peptide LFRQFY (derived from αs1-casein) were found to have highly inhibition potency with IC50 values of (7.9±1.7) μmol/L and exhibiting mixed inhibition patterns. The molecular docking results revealed that LFRQFY were mainly attributed to forming very strong hydrogen bonds with ACE amino acid residues Ala354 (S1 pocket) and His353 (S2 pocket)， and showed significant antihypertensive activity in vitro.