IgE-mediated hypersensitivity reaction to pollens is a common health problem in atopic patients. In this regard, the assessment of the allergenicity of highly pollinating plants would be demanding. Based on the increment of Ailanthus altissima (A. altissima) tree in some parts of Iran and considering its probable role in respiratory allergy, in this study, we aimed to investigate its IgE-immunoreactivity and in diagnostic applications. One hundred and twenty-five allergic rhinitis patients who were diagnosed as high IgE responders and demonstrated seasonal rhinitis or rhinoconjunctivitis, as well as 20 healthy controls (HCs) with no allergic symptoms, were enrolled in this study. Total protein extract was prepared from A. altissima pollens and subjected to quality control experiments and finally used in ELISA and western blotting studies. Approximately 24% of the atopic patients (30 from 125) showed positive immunoreactivity to A. altissima extract. The median (IQR) of absorbance (450 nm) of the specific IgE against A. altissima pollen extract in HCs and positive groups were 0.33 (0.28-0.42) and 0.59 (0.36-0.79), respectively (p<0.001). Receiver operating characteristics (ROC) curve analysis of the specific ELISA results, revealed a cut-off value of 0.46 and a sensitivity of 70% and specificity of 100%. Western blotting with the sera positive cases revealed that the main immunoreactive proteins range from 10 to 70 kDa. This study revealed that some of A. altissima pollen proteins ranging from 10 to 70 kDa show IgE-reactivity in atopic patients and may play a role in their allergic reaction symptoms.