The biological mechanism of adverse health outcomes related to exposure to traffic-related air pollution (TRAP) needs elucidation. We conducted a randomized, crossover trial among healthy young students in Shanghai, China. Participants wore earplugs and were randomly assigned to a 4-hour walking treatment either along a traffic-polluted road or through a traffic-free park. We conducted untargeted analyses of plasma exosome transcriptomics, serum mass spectrometry-based proteomics, and serum metabolomics to evaluate changes in genome-wide transcription, protein, and metabolite profiles in 35 randomly selected participants. Mean personal exposure levels of ultrafine particles, black carbon, nitrogen dioxide, and carbon monoxide in the road were 2-3 times higher than that in the park. We observed 3449 exosome mRNAs, 58 serum proteins, and 128 serum metabolites that were significantly associated with TRAP. The multi-omics analysis showed dozens of regulatory pathways altered in response to TRAP, such as inflammation, oxidative stress, coagulation, endothelin-1 signaling, and renin-angiotensin signaling. We found that several novel pathways activated in response to TRAP exposure: growth hormone signaling, adrenomedullin signaling, and arachidonic acid metabolism. Our study served as a demonstration and proof of concept on the evidence that associated TRAP exposure with global molecular changes based on the multi-omics level.