The electro-mechanical behaviors of transparent conductive oxide film on polymer substrate are of great concern because they would greatly affect the stability and lifespan of the corresponding devices. In this paper, indium tin oxide (ITO) films with different thicknesses were deposited on a polycarbonate (PC) sheet; meanwhile, in situ electrical resistance, in situ scanning electron microscopy and profilometry were employed to record the electrical resistance, morphologies and residual stress in order to investigate the fracture behavior and electrical-mechanical properties of ITO films under uniaxial tension loading. The electrical resistance changes, crack initiation, crack propagation and crack density evolution of ITO films were systematically characterized by in situ tests. Three fracture stages of ITO films were summarized: Ⅰ crack initiation, Ⅱ crack propagation, Ⅲ crack saturation and delamination. The crack initiation and electrical failure in a thinner ITO film occurred at relatively higher applied tensile strain; namely, the ductility of the film decreased as the film thickness increased. Residual compressive stress was recorded in the ITO films deposited on PC at room temperature and increased as the film thickness increased. Intrinsic crack initiation strain (CIS*) showed an opposite thickness dependence to residual strain (εr); the increase in residual compressive strain was counteracted by the decrease of intrinsic cohesion, leading to an overall decrease in effective crack initiation strain (CIS) when the film thickness increased. In addition, integrated with a formulated mechanics model and the analysis of the three fracture stages under tension, the fracture toughness and interfacial shear strength were quantitatively determined. As the film thickness increased (in the range of 50~500 nm), the fracture toughness decreased and the films were more prone to crack, whereas the interfacial shear strength increased and the films were less likely to delaminate.