When a micro-milling tool has a clamping angle on its spindle, the wear of the tool edge will accelerate and shorten the tool’s lifespan. In order to accurately observe the inclination state of the micro-milling tool on the machine, a three-dimensional pose reconstruction method based on the depth of field of a micro-milling tool is proposed. The laser coaxial digital holographic experimental device is used to obtain the micro-milling tool hologram, and the reconstruction image is obtained through the Fresnel reconstruction algorithm. The tool edge points are extracted as the key points in the reconstruction image, the wavelet transform local variance operator is used to obtain the degree of focus of the key points, and then the axial position corresponding to the milling tool is determined. The least square method is used to fit the key points and correct the reconstruction error, from which the three-dimensional pose reconstruction of the micro-milling tool is realized. The experimental results show that the reconstruction error of the micro-milling tool obtained by the three-dimensional pose reconstruction method is better than 0.1°. This method can accurately measure a three-dimensional pose of a micro-milling tool, which can provide a reference for the subsequent correction of micro-milling tool clamping accuracy.