Tunnels are the preferred experimental environments for estimating vehicle emission factors (EFs) under real-world driving conditions. In this study, online measurements of traffic-related air pollutants (including CO2, NOX, SO2, O3, particulate matter [PM], and volatile organic compounds [VOCs]) were conducted using a mobile laboratory in the Sujungsan Tunnel in Busan, Korea. Mobile measurements generated concentration profiles of the target exhaust emissions inside the tunnel. These data were used to produce a zonation of the tunnel, i.e., mixing and accumulation zones. There were differences between the CO2, SO2, and NOX profiles, and a starting point that was free from ambient air mixing effects could be set at 600 m from the tunnel entrance. The EFs of vehicle exhaust emissions were calculated using pollutant concentration gradients. The average EFs for CO2, NO, NO2, SO2, PM10, PM2.5, and ∑VOCs were 149,000, 380, 55, 29.2, 9.64, 4.33, and 16.7 mg km−1·veh−1, respectively. Among the VOC groups, alkanes contributed more than 70% of the VOC EF. Mobile measurement-derived EFs were validated using the conventional EFs from stationary measurements. The EF results from the mobile measurements matched those from the stationary measurements, while the absolute concentration differences between them implied complex aerodynamic movements of the target pollutants inside the tunnel. This study demonstrated the usefulness and advantages of applying mobile measurements in a tunnel environment and indicated the potential of the approach for observation-based policymaking.