β-lactoglobulin (BLG) is the primary allergen in bovine milk allergy. The identification of key amino acids in the BLG epitopes has not been comprehensive due to differences in identification methods, patient symptoms, and population characteristics. In this study, bioinformatics predictions were conducted for key amino acids based on two potential IgE linear epitopes in BLG. Then, the peptide AA30-43 was confirmed as an IgE linear epitope through alanine scanning mutagenesis and peptide microarray assays, with four key amino acids (A