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, $Ar/O_2$ 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 $Ar/O_2$ 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 $Ar/O_2$ 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 $6.44{ imes}10^{-4}{Omega}-cm$ 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.