Ultraviolet (UV) light is widely used for disinfection in indoor environments. Some wavelengths of UV light can produce high concentration of O3. UV irradiation combined with O3 may have great potential for nitration of allergens in the presence of NO2 in the air. In this study, the effects of UV irradiation on the nitration of three major indoor allergens including group Ⅰ allergens of house dust mite (Der p 1 and Der f 1) and group Ⅰ allergen of dog (Can f 1) in the presence of NO2 and O3 were investigated by analysis of the protein quantity, tyrosine, peptides, and nitration degree. The results showed that UV irradiation induced a significant increase in the quantity of 3-nitrotyrosine in the allergens from 0.4 ± 0.4 ng to 4.0 ± 0.8 ng. After 12 h of UV-O3 co-exposure, the total nitration degrees of the three allergens ranged from 0.1% to 0.5%, which were significantly higher than those after only O3 exposure (p < 0.05). The analysis of peptides revealed that the nitration of tyrosine was site-specific. The tyrosine Y231, which was adjacent to aspartic acid, posed the highest nitration degree of 41.1 ± 24.0% in Der p 1. The nitration degree of tyrosine Y162 was the highest (1.7 ± 0.1%) in Der f 1. Overall, this study demonstrated that UV irradiation enhanced the O3-related nitration of allergens in the air, which provides an experimental basis for the impact of daily disinfection behavior on allergens.