Waste silk as raw material was dissolved with 40% CaCl₂ solution at first, and silk fibroin was extracted after dialysis; Then the enzymolysis conditions of silk fibroin was optimized. Besides, silk fibroin peptides was fractionated by using HPGFC and separated ulteriorly by RP-HPLC, then their anti-alcoholism activity in vitro was detected by Valle＆Hoch’s method. The experiment results showed that the optimal enzymatic hydrolysis conditions of silk fibroin were as followed: time of 240 min, enzyme dosage of 2%, substrate concentration of 5%, pH 8.5 and temperature of 60 ℃, and the optimal degree of hydrolysis（DH）was 23.22%. In addition, the better alcohol dehydrogenase (ADH) activation rate of SFP-II-8 obtained by HPGFC and RP-HPLC was measured under the concentration of 2 mg/mL, and it can up to 44.51%. UPLC-Q-TOF-MS analysis showed that its amino acid sequence was L-G-G-V-G-A.