The invention discloses an automatic focusing method of an electron microscope. The method comprises the following steps of initializing a stepping motor, arranging the stepping motor at an initial position, acquiring a first picture; performing full-picture processing on the first picture, obtaining t areas with most obvious image changes, and outputting gradient outburst values of t areas; after one picture is processed, calculating and outputting t gradient outburst values, and frontwards moving the stepping motor by pi step lengths each time until the whole stroke is finished; respectively fitting all gradient outburst values and corresponding motor positions in the t areas for forming t secondary curves, finding out a motor position which corresponds with a peak value from the secondary curves, wherein the position is the focal length which corresponds with a highest-definition image; moving the stepping motor to the position for finishing focusing, and simultaneously acquiring the image and displaying. The automatic focusing method can eliminate the effect of different ambient lights and different photographed object material surfaces on the automatic focusing effect of the electron microscope.