The phage display technique has been a useful tool for selecting and developing inhibitors of enzymes that are of significance for pharmacology and medicine. In this study, a phage display peptide library was screened against a food enzyme, plasmin, to develop a specific and novel peptide inhibitor. The peptides inhibiting the target enzyme were modified by deleting preferential cleavage sites for plasmin. The inhibitory constant (Ki) of the peptide having highest inhibitory effect was found to be 207 μm. The binding of the peptide to plasmin was tested with isothermal titration calorimetry and the affinity constant was measured as 105 m−1. The selected peptide was highly specific for plasmin, as it showed no inhibitory effect on trypsin. Finally, the inhibitor activity of the peptide in milk sample was investigated, and the developed peptide was concluded to be a promising inhibitory agent against plasmin to be used in food applications.