A novel measurement scheme for the stabilization accuracy of a Line of Sight(LOS) in an electro-optical imaging system on a moving carrier is proposed. The principle of optical measurement for LOS stabilization accuracy is briefly introduced, and the optical design of an autocollimator with a large field of view and a detection algorithm for the light-spot position are investigated.The autocollimator is used to test orientation changes of the LOS which is represented by a mirror fixed on the frame of the electro-optical imaging system.A high-speed CMOS camera for the collection of images and an algorithm for image registration based on Phase-only Correlation (POC) are employed to detect sub-pixel level displacement of light-spot images by calculation of the normalized cross-power spectrum between pairs of images. The scheme has been used in the LOS stabilization accuracy measurement system, and a calibrating experiment is set up using a Leica theodolite. The experimental results show that the RMS error of the angular measurement is less than 2 μrad when the field of view is ±1° and the sampling rate is 500 frame/s. The dynamic measurement can meet the system requirements of a larger field of view and a greater precision.