Chinese shrimp (Penaeus chinensis) is widely cultured and consumed in Asia, but is also a major food allergen locally. Though to be the foundation of preventing and treating allergy, the allergenic epitopes of major allergens tropomyosin (TM) and arginine kinase (AK) in Penaeus chinensis have not been identified. Here, we applied Immunoinfo-CB (immunoinformatics coupled with competitive-binding strategy) to address the point. Potential allergenic epitopes of TM and AK were predicted by multiple immunoinformatics tools, followed by validating with inhibitory dot-blot assay, indirect competition ELISA and mast cell degranulation assay. Furthermore, critical amino acids in allergenic epitopes were also identified by Immunoinfo-CB. Our findings provide new insight into allergenic epitopes and critical amino acids of TM and AK responsible for the anaphylactic response. The Immunoinfo-CB therefore offers promises for characterization of IgE-binding epitopes that might be used as new targets for immunotherapy of food allergy.