A new method of uncertainty evaluation of centroid detection is proposed. The uncertainty associated with the intensity of a pixel, belonging to the target to be detected, is firstly evaluated from the pixel intensity and the image gradient at the pixel. Then the uncertainties associated with the centroid detector are evaluated according to the relation between the evaluation of pixel intensity uncertainty and the pixel. The mathematical model to evaluate the uncertainty of pixel intensity and the analytical expression to evaluate the uncertainty of centroid detection are presented. The experimental results show that the uncertainty of centroid detection can be evaluated accurately with this method.