Shellfish has been recognized as one of the leading causes of food allergy in both adults and children in Asia Pacific region. In Malaysia, black tiger prawn (Penaeus monodon) is among the most widely consumed species. Our previous studies have successfully identified several major allergens including a thermostable protein of 36 kDa. Thus the aim of this study was to identify the 36 kDa major allergen by an allergenomic approach. Protein extracts of raw prown were prepared and resolved by 2-dimensional electrophoresis (2-DE). Immunoblotting was then performed using sera from patients with prawn allergy. Selected spot from 2-DE was then excised, digested and analyzed by mass spectrometry. The 2-DE profile of the extract revealed approximately 100 protein spots between pH of ~4 to 10 and the size range between (<10 to 250 kDa). The 2-DE immunoblotting has detected numerous IgE-binding spots at 36 kDa. Mass spectrometry analysis of the major IgE-binding spot (spot 1a) has identified the 36 kDa spot as tropomyosin. Our findings indicated that tropomyosin play a major role in allergic reaction to black tiger prawn among local patients with prawn allergy, and should be included in diagnostics and therapeutic strategies of this allergy.