Due to transmission backlash of precise cable drive in electro-optical targeting system, it is difficult to predict force and pointing precisely during target detection. This article aims to develop a rigorous model for predicting the cable tension and transmission backlash of precise cable drive in electro-optical targeting system to better evaluate the performance in the preliminary design stages. The model of transmission backlash in different phase is established by satisfying the system’s geometric and equilibrium constraints, which is applicable to cable drive systems with any external load, pretension, and motion trajectories. Simulations are conducted to reveal the changes of cable deformation, cable tension, and deflection angles. The model is validated by experiments using a suitable experimental setup, predicting transmission backlash with the error level of 8.27%. The sensitivities of the preload force and external load are tested, and corresponding results are in good agreement with the theoretical ones. The backlash plunges dramatically when the preload force is small and flattens out when the preload force is larger than the threshold value. A proper preload force should be applied to keep the backlash in stable state. The threshold value could be predicted by the proposed model. The method presented in this article could provide a theoretic guide for system design and nonlinearity compensation.