The main objective of the current study is to establish an on-chip strategy to analyze the allergens existing in the myofibrillar proteins of seafood matrices. On-chip immunomagnetic separation (IMS) of allergens derived from large yellow croaker (Larimichthys crocea), swimming crab (Portunus trituberculatus) and black tiger shrimp (Penaeus monodon), as model seafoods, was studied. The analysis of the tryptic digests of on-chip captured proteins, combined with protein database searches, was performed for the identification of allergens by MALDI-TOF MS with a 0% false-positive rate. By using the sera of ten seafood-allergic patients, six allergens were identified from the myofibrillar proteins of the large yellow croaker. The present protocol was successfully validated by ELISA and can be applied in blood tests for food allergies with a single drop of blood. The current protocol is presumed to be applied in the identification of potential allergens in other organisms.