Background Perilla seeds have been shown to cause immediate allergic reactions. However, reports on perilla seed allergies are limited to a few case reports, and there is currently no diagnostic test for such allergies. 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-two children with clinical perilla seed allergy were enrolled from two tertiary hospitals between September 2016 and June 2019. Using perilla seed extract, we developed a skin prick test (SPT) reagent and an IgE enzyme-linked immunosorbent assay (ELISA) for perilla seed allergy diagnosis. IgE immunoblotting was performed for identifying 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, and the proportion of children with anaphylaxis was 31.8%. Perilla seed SPT was performed for 16 of 22 children, all of whom tested positive. On ELISA, 86.4% of children tested positive for perilla seed-specific IgE. Proteins with molecular weights of 50, 31–35, and 14–16 kDa showed binding with the sera of >50% of children with perilla seed allergy. LC-MS/MS analysis of these three protein fractions indicated 8 putative proteins, including perilla oleosin (Accession No. 9963891), to be allergens. Conclusion We reported the clinical characteristics and immunological profiles of 22 children with perilla seed allergy and suggested oleosin as one of the major allergens in perilla seeds.