Tropomyosin (TM), known as the major allergen in seafood, has been increasing concerns in industrialized countries. We proposed a simple, rapid and accurate analytical method using surface plasmon resonance (SPR) sensor with gold patterned biochips for detection and quantification of TM in shellfish. The limit of detection (LOD) was 1.0 μg mL−1 (S/N = 3), while the limit of quantitation (LOQ) was 2.5 μg mL−1 (S/N = 10). Moreover, the satisfactory recoveries (92.0–108.0%) was obtained for TM detection in food samples, showing the good accuracy of this method. Furthermore, the results also demonstrated that this developed method is suitable for the determination of TM from different shellfish species within 3 min. Therefore, the developed SPR sensor with gold patterned biochips will have positive significance on the rapid and accurate detection allergens in shellfish foodstuffs, related products and their production lines in the future.