The oxidation of sulfur dioxide (SO₂) to sulfur trioxide (SO₃) is an undesirable reaction that occurs during the selective catalytic reduction (SCR) of nitrogen oxides (NO_x) with ammonia (NH₃), which is a process applied to purify flue gas from coal-fired power plants. The objectives of this work were to establish the fundamental kinetics of SO₃ formation over a V₂O₅/TiO₂ catalyst and to illustrate the formation mechanism of SO₃ in the presence of NO_x, H₂O and NH₃. A fixed-bed reactor was combined with a Fourier transform infrared (FTIR) spectrometer and a Pentol SO₃ analyser to test the outlet concentrations of the multiple components. The results showed that the rate of SO₂ oxidation was zero-order in O₂, 0.77-order in SO₂ and -0.19-order in SO₃ and that the apparent activation energy for SO₂ oxidation was 74.3 kJ mol⁻¹ over the range of studied conditions. Based on in situ diffuse reflectance infrared Fourier transform (in situ DRIFT) spectroscopy, X-ray photoelectron spectroscopy (XPS) and temperature programmed desorption (TPD) tests, the SO₃ formation process is described here in detail. The adsorbed SO₂ was oxidized by V₂O₅ to produce adsorbed SO₃ in the form of bridge tridentate sulfate, and the adsorbed SO₃ was desorbed to the gas phase. NO_x promoted the oxidation of the adsorbed SO₂ due to the promotion of the conversion of low-valent vanadium to high-valent vanadium. In addition, the desorption of the adsorbed SO₃ was inhibited by H₂O or NH₃ due to the conversion of tridentate sulfate to the more stable bidentate sulfate or ammonium bisulfate. Finally, the mechanism of the influence of NO_x, H₂O and NH₃ on the formation of gaseous SO₃ was proposed.