Sesame is one of the eight major allergens that cause food allergies. Study of the epitopes of sesame allergens is important for understanding their sensitization mechanisms. Currently, less information is available on the epitope studies of sesame allergens. In this study, we analyzed the molecular characteristics, structure and homology of Ses i 3, one of the important sesame allergens. We predicted the B-cell linear epitopes of Ses i 3 using bioinformatics tools and characterized them by slot blot immuno-microarrays technology. Eight peptides as B-cell linear epitopes of Ses i 3 were identified, in addition, key amino acids in these epitopes were predicted and leucine 422 was identified as a key amino acid. The present work will contribute to further understanding of the sesame allergen and provide some help in the prevention and treatment of sesame allergy.