Alpha-lactalbumin (ALA) is one of the major allergens in cow’s milk. However, research on its conformational epitopes has been relatively limited. Antibodies against cow’s milk ALA was obtained from eight children by two-step affinity chromatography. Subsequently, mimotopes against IgG and IgE were biopanned from phage display libraries, respectively. Based on the mimotopes, continuous epitopes were defined according to the peptide mimic frequency determined with the UniProt alignment tool.