The line-of-sight (LOS) stabilization control is required to isolate LOS from the movement and vibration of carrier and ensure pointing and tracking for target in electro-optical tracking system. In this paper, a fractional-order PI (FOPI) controller is applied in the design of stabilization loop of LOS stabilization system based on the analysis of the fractional calculus theory. This controller exhibits excellent performance even in presence of nonlinearity and uncertainty. Details of this controller along with the performance comparisons between FOPI and conventional integer-order PI (IOPI) are presented. Simulation results indicate that the FOPI controller can improve the carrier disturbance rejection performance of LOS stabilization system.