Crustacean is one of the major allergic foods. It is of great significance to identify more crab allergens and research the detection methods for crab food anaphylaxis. In this study, IgE reactivity to three recombinant proteins from Portunus trituberculatus, including tropomyosin (rPtTM), myosin light chain (rPtMLC), and pancreatic lipase (rPtPL), were detected by enzyme-linked im- munosorbent assay (ELISA). The expressions of tropomyosin (TM) in various tissues of P. trituberculatus were detected by Western blot (WB). Furthermore, microplates were coated with rPtTM and the ELISA conditions were optimized. The cut-off value was deter- mined by the receiver operating characteristic (ROC) curve. Among 51 crab-allergic sera, 21 (41.2％) showed positive IgE to rPtTM. Other 70 crab-allergic sera more frequently recognized rPtPL (9/70; 12.9％), followed by rPtMLC (1/70; 1.4％). WB results showed that TM was mainly expressed in the muscle, followed by the heart and a small amount in gills and the testis. The optimal results showed that the coating condition of rPtTM was 50 ng per well with coating for 3 h at 37℃. The optimal blocking condition was 1.2％ BSA with blocking for 3 h at 37℃, and the optimal dilution of the second antibody was 1:1500. The ROC curve showed that the ELISA reagent had high sensitivity (83.52％) and specificity (98.00％) when the cut-off value was 0.45. All results indicated that tropomyosin is the major allergen of P. trituberculatus, and myosin light chain and pancreatic lipase are the potential allergens. Addi- tionally, the ELISA reagent developed with the rPtTM was feasible for laboratory detection of crab anaphylaxis.