The presence of SO3 severely affects not only the safe and economic operation of coal-fired power plants but also the atmospheric environment. The purpose of this study was to investigate the generation of SO3 over V2O5-WO3/ TiO2 catalysts. The SO3 generated during the reaction was collected by the controlled condensation method. N-2 adsorption, X-ray fluorescence (XRF), Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), SO2 temperature-programmed desorption (SO2-TPD), and in situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) experiments were performed to evaluate the catalyst properties. The experimental results show that the generation of SO3 on the catalyst is complicated and can be promoted by NO and NH3. The generation rate of SO3 increased from 0.5 vol% to 3.29 vol% with an increase in the NO concentration from 0 to 1000 ppm. When 500 ppm NH3 was added to an atmosphere containing 500 ppm NO, the generation rate of SO3 increased from 2.23 vol% to 4.23 vol%. The addition of NH3 led to a significant reduction in the specific surface area of the catalyst due to the generation of ammonium bisulfate (ABS). In addition, the results show that the proportion of lattice oxygen (O alpha) and the adsorption of SO2 on the catalyst would be promoted by NO and NH3, and DRIFTS experiments further proved that NO and NH3 promoted the generation and transformation of the intermediate products VOSO4 and HSO4-.