This study compared and analyzed the toxic concentration and impact range that could kill 50% of the people in the Samae 2 Tunnel by using the ALOHA PROGRAM and Probit analysis methods for each substance, including ammonia, in the event of an accidental gas leak at the exit of a Level 3 road tunnel while transporting toxic substances in tanks fixed to vehicles, and applied the results to Google Earth. This study showed that the impact range differs by substance when toxic gases stay and move in the tunnel. Therefore, it is necessary to change the direction of installing additional or reducing evacuation connection routes by referring to the impact range using simulations for each substance. The results of this study estimated that there is a high probability of 50% of casualties due to toxic concentrations, so it is necessary to recognize that toxic gases in tunnels also pose a potential risk of casualties just like smoke, and in the future, it is necessary to establish new standards for smoke extraction or exhaust to expel toxic gases out of tunnels.