The invention discloses a positioning device and a positioning method of a scanning electron microscope sample stage. The positioning device comprises a camera, an image collection system adjusting bracket, an image collection system base station, a sample stage base, a positioning sample stage, an image controller and a data processing and outputting system. Through introducing the positioning sample stage and combining with image collecting and processing processes, positioning of different sample positions on the sample stage is achieved by a scanning electron microscope sample stage coordinate calibration method; the complicated and error-prone processes of searching and positioning samples through small-scale movement of the sample stage within a small view of the scanning electron microscope are omitted; accurate and rapid positioning of the samples is achieved; the experiment efficiency of the scanning electron microscope is greatly improved; and a lot of time and cost for searching and positioning the samples can be saved for a scanning electron microscope user.