Various damages, including corrosion, fouling, and blue plumes, can be caused by sulfur trioxide (SO3) in the flue gas of a coal-fired boiler on boiler equipment and the atmospheric environment. The increasing demand for energy conservation and emission reduction requires accurate knowledge on predicting and controlling the SO3 concentration. Accurate knowledge on the effect of the factors that influence SO3 in flue gas is necessary as well. Therefore, in this study, various SO3 concentrations under different flue gas conditions are measured with measuring devices built based on controlled condensation and S balance methods. To further study the effect of the factors that influence SO3 in flue gas, an improved SO2/O-2/H2O/CO2/CO/NO kinetic mechanism is built based on previously developed mechanisms and validated with previous experimental data. SO3 concentrations under the established flue gas conditions and with increasing residence time are numerically calculated. The SO3 concentration in flue gas is mainly dependent upon SO2, O-2, and H2O concentrations as well as the temperature and residence time. The SO3 concentration undergoes three stages of sharp increase, slow increase, and gradual decrease as the residence time increases. In addition, the SO3 concentration increases significantly as CO and NO emerge in the flue gas. However, SO3 is constant as CO2 emerges.