Indium tin oxide (ITO) films had been deposited on glass substrate without substrate heating and then tungsten oxide (WO3) films were deposited on ITO films by DC magnetron sputtering. In this work, we present the annealing ambient effect of ITO substrate on electrochromic properties of WO3 films. The ITO films were annealing in air and in vacuum at 350°C before coating with WO3 films. The structural, optical, and electrical properties of ITO films for as-deposited, annealing in air and in vacuum were investigated by X-ray diffraction, UV-VIS-NIR spectroscope and four point probe. The ITO films had a better crystallinity and lager grain size after annealing in air and in vacuum. The resistivity of ITO films increase with annealing in air, but decrease with annealing in vacuum. The WO3 films show difference surface morphology with higher grain size and surface roughness when coating on annealed ITO films in both cases. The electrochemical properties of film systems were characterized by cyclic voltammetry. The film systems of ITO plus WO3 showed that the charge capacity of ITO substrate annealing in vacuum was higher than the as-deposited ITO substrate and the ITO substrate annealing in air, respectively. This result corresponded to electrical conductivity of each ITO substrate.