Aspergillus terreus has been described as an allergenic fungus in addition to causing infection to both human beings and plants. However, the allergens in this fungus are still unknown. The identification of allergens is essential to develop diagnostic and therapeutic avenues. Employing a proteomic approach, sixteen allergens were identified based on two-dimensional immuno-blotting with A. terreus susceptible patient sera. Amongst them, triosephosphate isomerase (Asp t 36), one of the dominant immunoglobulin E (IgE)-reactive proteins, was purified under native conditions. To characterize Asp t 36, the gene was cloned and expressed in E. coli. Phylogenetic analysis showed it to be highly conserved with close similarity to Dermatophagoides farinae. Four immuno-dominant epitopes were identified using synthetic peptides and mapped on the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure rendering it to be an IgE binding hotspot. Asp t 36 showed similar secondary structure and temperature sensitivity with other reported triosephosphate isomerase allergens. In-vivo studies using the murine model demonstrated rAsp t 36 able to stimulate airway inflammation, such as an influx of eosinophils, goblet cell hyperplasia, elevated serum immunoglobulins and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus for the first time in any fungi. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.