The accurate positioning of laser spot center plays an important role in confocal microscopy system. In order to improve the accuracy of spot center location, a high-precision spot center location detection method based on OpenCV is designed. First, the denoise and filter the collected spot image, and then filter the connected domain to eliminate gross errors through clustering analysis. Then, perform morphological processing on the edge of the connected domain of the object, and finally fit the edge data into an ellipse to locate the spot center. The simulation and experimental results show that the method in this paper has a sub-pixel level positioning accuracy of less than 0.1 pixel and more stable detection results than other traditional spot center location methods, and is a high-precision spot center location method.