The electrical, optical, structural and surface properties of an indium tin oxide (ITO) film grown on a flexible PET substrate using a specially designed roll-to-roll (R2R) sputtering system as a function of the DC power, Ar/O2 flow ratio, and rolling speed is reported. It was observed that both the electrical and optical properties of the ITO film on the PET substrate were critically dependent on the Ar/O2 flow ratio. In addition, x-ray diffraction examination results showed that the structure of the ITO film on the PET substrate was an amorphous structure regardless of the DC power and the Ar/O2 flow ratio due to a low substrate temperature, which was maintained constant by a main cooling drum. Under optimized conditions, ITO film with resistivity of 6.44×10-4 Ω-cm and transparency of 86% were obtained, even when prepared at room temperature. Furthermore, bending test results exhibited that R2R-grown ITO film had good flexibility which would be applicable to flexible displays and solar cells.