Abstract: Spot center location is a key technique in the field of optical measurement. A Two-step method for center location of small-scale focal spot with high precision was proposed to solve the problem as the low precision when using classic center location algorithms. First, the pixel-level center could be located through zero crossing of the first derivative in the focal spot image. And then, the unsaturated part of the focal spot beside the pixel-level center was used to fit the Gaussian surface to calculate the sub-pixel center. Experimental results show that, when comparing with other classic center location algorithms, the error of the two-step method is much less than 0.05 pixels in the spot image without background noise. The two-step method can keep high-precision center location of the focal spot. If the imaging model of the focal spot is much more similar with the ideal Gaussian spread function, the precision is higher.