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.