Misgurnus anguillicaudatus(loach) is a common freshwater fish in Asia， which has higher nutritional and medical value. In this study， the loach was hydrolyzed with protease secreted by Bacillus pumilus 4SP5 and the hydrolysis products of different molecular weights were obtained by ultrafiltration. Among them， the fractions smaller than 5 ku showed the strongest antioxidant activity. The filtration components were purified by G25 chromatography filtration and sequenced and identified by LC-MS/MS， and 6 potential bioactive peptides were obtained. Among them， the oligopeptide AFRVPTP had good DPPH scavenging activity (IC50 37 μg/mL)， hydroxyl radical scavenging activity， and superoxide anion scavenging activity (IC50 0.73 mg/mL). The oligopeptide was also shown to inhibit α-glucosidase with an IC50 of 15.86 mg/mL and was predicted to be non-toxic and non-sensitizing online. The results of molecular docking showed that AFRVPTP acted by binding key amino acids in the catalytic domain of the enzyme. It could interact with key amino acids residue of the active domain of Keap1， which indicated that the AFRVPTP had the application potential to regulate the antioxidation (Asn387， Arg415， Tyr334， Arg380， Gln530， Tyr525， Arg483， Ser508) in vivo. The results provided experimental basis for the intensive processing of loach.