Food proteins undergo significant structural changes during various processing methods
however, key epitopes often exhibit resistance to damage and modification. Recently, we developed a sensitive sandwich enzyme-linked immunosorbent assay (Epi-mAbs-sELISA) based on IgE linear epitope peptides. This method enables the quantitative detection of alpha-lactalbumin (ALA) for the assessment of food allergenicity. We determined that antibodies developed from IgE linear epitopes possess higher affinity. Ultimately, the optimized Epi-mAbs-sELISA presented in this study demonstrated a wide linear detection range (30.52 ng/mL to 125 μg/mL), a low detection limit (1.122 ng/mL), and excellent precision, with an internal determination relative standard deviation of 9.23% and an external determination relative standard deviation of 9.89%. The recoveries across various food matrices ranged from 81.9% to 111.9%, surpassing the sensitivity and detection range of commercial ELISA kits. The application of this testing method to processed food samples confirmed its ability to detect undeclared ALA residues, thereby mitigating potential safety issues for patients with milk allergies.