To study the influence of the light source, object, measuring system and subpixel location algorithm on the sub-pixel locating accuracy, a CMOS imaging system is designed for experiment and data treatment. The results show that the stability of the source and the measuring surroundings have notable effect on the high accuracy of measurements. The improved centroid algorithm with a threshold has the same effect on de noise but can reduce the interference signal dramatically and especially with a dynamic threshold while the interference signal has the property of slight change. Choosing an algorithm appropriately according to the real object and the error distribution is the key to gain the high precision subpixel location in engineering application.