Abstract: In order to investigate the optimal extraction process and anti-inflammatory activity of white kidney bean polypeptides (WKBPs), this study employed white kidney bean protein as raw material and hydrolyzed white kidney bean protein by ultrasound-assisted enzymatic hydrolysis, utilizing the inhibition rate of bovine serum protein degeneration as the index. We identified the optimal extraction process with a single-factor combined response surface method and separated the prepared white kidney bean crude peptides with ultrafiltration tubes of different molecular weights. This study determined the anti-inflammatory activity of various components in vitro, detected the optimal components by liquid chromatography-mass spectrometry (LC-MS/MS), and examined the anti-inflammatory mechanism by molecular butt grafting technique. The results indicated that the inhibition rate of WKBPs on bovine serum protein denaturation was 86.57% under the conditions of 166 W ultrasonic power, 18000 U/g enzyme addition, 128 min enzymatic hydrolysis time, and 46 ℃ enzymatic hydrolysis temperature. After ultrafiltration, the components with Mw<1 kDa revealed good anti-inflammatory activity (bovine serum protein denaturation inhibition, hyaluronidase inhibition, and NO inhibition), and the IC50 values were 1.181, 9.244 and 10.887 mg/mL, respectively. We detected 3306 peptides by LC-MS/MS, of which 73% were peptide sequences ranging from 3 to 10. Virtual screening Vina score with type and induced nitric oxide synthase (inducible nitric oxide synthase, iNOS) binding energy minimum peptide sequence of 4 peptide FFFR, Molecular docking visualization findings demonstrated that 4-peptide FFFR can bind to the active site of iNOS via hydrogen bonding and hydrophobic interaction, thus exerting inhibitory effects. This study serves as a reference for further research on the anti-inflammatory activity of WKBPs.