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Food-derived peptides exhibit potential immunomodulatory effects and become popular functional ingredients in food industry. However, the in vivo effect varies from in vitro while the optimal utilisation content remains unclear. This study aimed to identify novel peptides from protease-facilitated fermentation, to evaluate their immunomodulatory effect and effect-dose relationship in vivo. Totally 2045 peptides were screened by liquid chromatography mass spectrometry (LC-MS/MS), and in silico methods were applied to predict the potential bioactivity through Peptide Ranker, GRAVY Calculator and PepDraw. In vivo assay indicated that 200 mg kg −1 milk-derived peptides showed the optimal regulation on lipopolysaccharide-induced inflammation by enhancing the immune function of spleen and regulating immunoglobulin and cytokine production. These peptides contributed to the prevention of inflammation through the oral route and could be further applied in functional food development.