Transportation emissions significantly affect human health, air quality, and climate in urban areas. This study conducted experiments in an urban tunnel in Taipei, Taiwan, to characterize vehicle emissions under real driving conditions, providing emission factors of PM2.5, eBC, CO, and CO2. By applying multiple linear regression, it derives individual emission factors for heavy-duty vehicles (HDVs), light-duty vehicles (LDVs), and motorcycles (MCs). Additionally, the oxidative potential using dithiothreitol assay (OPDTT) was established to understand PM2.5 toxicity. Results showed HDVs dominated PM2.5 and eBC concentrations, while LDVs and MCs influenced CO and CO2 levels. The CO emission factor for transportation inside the tunnel was found to be higher than those in previous studies, likely owing to the increased fraction of MCs, which generally emit higher CO levels. Among the three vehicle types, HDVs exhibited the highest PM2.5 and eBC emission factors, while CO and CO2 levels were relatively higher for LDVs and MCs. The OPDTTm demonstrated that fresh traffic emissions were less toxic than aged aerosols, but higher OPDTTv indicated the impact on human health cannot be ignored. This study updates emission factors for various vehicle types, aiding in accurate assessment of transportation emissions' effects on air quality and human health, and providing a guideline for formulating mitigation strategies. Display omitted •Evaluated emission factors for heavy-duty, light-duty vehicles and motorcycles.•Quantify potential toxicity of fleet emissions using oxidative potential.•Motorcycles could contribute significantly to elevated CO emissions in urban areas.•Fresh traffic emissions show less toxicity than aged aerosols but still pose risks.•Updated emission factors guide mitigation strategies for air quality and health.