Abstract: The present disclosure provides a machine vision-based automatic focusing and automatic centering method and system, which belongs to the technical field of machine vision-based automatic control. An object stage is controlled to move in an imaging distance range of an electron microscope, and images scanned by the electron microscope when the object stage is at different imaging distances are acquired. The image definition of an object stage image is calculated according to a gray-scale value of each pixel in the object stage image, the imaging position when an image definition value is the maximum is determined, and the object stage is controlled to move to the position, so as to realize machine vision-based accurate focusing.