Rapidly monitoring and accurately assessing the direct impacts of air pollution on human health is a long-standing challenge. Exhaled breath analysis, as a real-time and sensitive human biomonitoring technique, is causing significant concerns. This study aims to analyze and assess volatile organic compounds (VOCs) in human exhaled breath under ambient ozone (O3) exposure. A comprehensive correlation analysis involving 288 VOCs are identified in the exhaled breath of healthy adults (n = 1130), uncovering 12 VOCs including methanol, propyne, and 1-butene etc. that show strong exposed correlations with ambient O3 levels. Nonlinear modeling indicated the dose-response commencing at O3 concentrations as low as 58 μg/m3. Moreover, the presence of PM2.5 significantly exacerbates the effect of O3 on the filtered 12 VOCs. The most pronounced interplay is observed at 140 μg/m3 for O3 and 150 μg/m3 for PM2.5. Finally, an in-vivo study using rats validates the result, with all of the 12 exhaled breath biomarkers exhibit obvious differences (O3 exposure group vs control group). This work demonstrates direct evidence that ambient O3 pollution impacts on human exhaled VOCs, and also offers valuable insights for establishing O3 exposure thresholds.