Background: Perilla seeds are known to cause immediate allergic reactions. However, reports on perilla seed allergy are limited to a few case reports worldwide, and there is currently no diagnostic test for this allergy. Objective: Our objective was to analyze the clinical and immunological characteristics of perilla seed allergy and to identify allergens for the development of diagnostic methods. Methods: Twenty-one children with clinical perilla seed allergy were enrolled from 2 tertiary hospitals between September 2016 and June 2019. Using perilla seed extract, we developed a skin prick test (SPT) and an IgE enzyme-linked immunosorbent assay (ELISA) for diagnosis of perilla seed allergy. IgE immunoblotting was performed to identify putative allergenic components, and amino acid composition analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: The median age of children with perilla seed allergy was 3 years; the proportion of children with anaphylaxis was 28.6%. SPT was performed with perilla seed in 15 of 21 children, all of whom tested positive. On ELISA, 85.7% of children tested positive for perilla seed-specific IgE. Proteins with molecular weights of 50, 31-35, and 14-16 kDa bound to the sera of >50% of children with perilla seed allergy. LC-MS/MS analysis of these 3 protein fractions showed 8 putative proteins, including perilla oleosin (Accession No. 9963891), to be allergens. Conclusion: This study documented the clinical characteristics and immunological profiles of 21 children with perilla seed allergy. Our results suggest that oleosin is one of the major allergens in perilla seeds.