The oil extraction residue of walnuts is rich in proteins and has been employed in the formulation of various functional food products. In this study, alcalase and neutrase were used to hydrolyze defatted walnut meal protein to obtain anti-inflammatory peptides. After separation by ultrafiltration and by using Sephadex G-25, the fraction with the highest anti-inflammatory activity was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and 579 peptides were obtained. Then, four of the most stable binding tripeptides with the sequences Trp-Pro-Leu (WPL, MW: 414.2 Da), Trp-Ser-Leu (WSL, MW: 404.2 Da), Phe-Pro-Leu (FPL, MW: 375.2 Da) and Phe-Pro-Tyr (FPY, MW: 425.2 Da) were successfully identified by virtual screening. The anti-inflammatory activity determination of the synthetic peptide assay indicated that FPL (200 μM) exhibited excellent anti-inflammatory activity with inhibitory rates of 63.65 ± 2.64%, 68.25 ± 2.19%, 42.52 ± 2.01% and 59.39 ± 2.21% in terms of four inflammatory mediators (NO, TNF-α, IL-6 and IL-1β), respectively. It was speculated that the anti-inflammatory activity of walnut peptides might be related to hydrophobic amino acids and aromatic amino acids. By molecular docking, further insight into the theoretical interaction mechanism of binding revealed that hydrophobic interactions and hydrogen bonds turned out to be the main interaction forces between the four peptides and iNOS. These results indicated that FPL screened in this study could be expected to be used as a natural anti-inflammatory active substance in the functional food and pharmaceutical industries.