The invention belongs to the technical field of micro-imaging, and discloses an automatic focusing method for a scanning type slice imaging device. The method comprises the steps of slicing a sample by a machining operation module, adjusting an object lens in two dimensions by a two-dimension translation stage, forming real time images of a cutter by an imaging module, and collecting and recording an image of multiple images obtained continuously through a data collection module; during the first dimension adjustment process, performing a blade position scale calculation for the image obtained in real time; during the second dimension adjustment process, calculating a gray level co-occurrence matrix for the image of the multiple images obtained continuously; obtaining a contrast value of the image through a gray level co-occurrence matrix; and obtaining a maximum of the contrast value by contrasting the contrast values of the multiple images obtained continuously. According to the invention, an imaging focusing problem of a moving object is solved indirectly by focusing the cutter in static state.