A highly transparent and thermally stable polyimide (PI) substrate was prepared and used for the fabrication of indium tin oxide (ITO)/PI films via radio-frequency magnetron sputtering at an elevated substrate temperature. The effect of the deposition conditions, that is, the oxygen flow rate, substrate temperature, sputtering power, and working pressure, on the optical and electrical properties of the ITO/PI films were investigated from the microstructural aspects. The results indicate that the optical and electrical properties of ITO were sensitive to the oxygen. Moreover, it was beneficial to the improvement of the ITO conductivity through the adoption of a high substrate temperature and sputtering power and a low working pressure in the deposition process. A two-step deposition method was developed in which a thick bulk ITO layer was overlapped by deposition on a thin seed ITO layer with a dense surface to prepare the highly transparent and conductive ITO/PI films. The ITO/PI film after annealing at 2408C gave a transmittance of 83% and a sheet resistance of 19.7 X/square.