The quantitative risk assessment (QRA) is one of the explicit requirements under the European Union (EU) Directive (2004/54/EC). As part of this, it is essential to be able to estimate the number of fatalities in different accident scenarios. In this paper, a tangible methodology is developed to estimate the number of fatalities caused by toxic gases due to fire in road tunnels by incorporating traffic flow and the spread of fire in tunnels. First, a deterministic queuing model is proposed to calculate the number of people at risk, by taking into account tunnel geometry, traffic flow patterns, and incident response plans for road tunnels. Second, the Fire Dynamics Simulator (FDS) is used to obtain the temperature and concentrations of CO, CO2, and O2. By taking advantage of the additivity of the fractional effective dose (FED) method, fatality rates for different locations in given time periods can be estimated. An illustrative case study is carried out to demonstrate the applicability of the proposed methodology.