Abstract: Digital Holographic Microscopy (DHM) combines optical interferometry with optical microscopy and hence provides a fast, non-destructive measurement approach for the 3D profiles of reflective samples, or the thickness distributions and refractive index distributions of transparent objects. DHM can automatically refocus the sample under inspection through numerical propagation of the object wave, if the defocusing distance is pre-known. Hence, it has always been being a hotspot in DHM research to automatically determine the defocusing distance. To this end, this paper reviewed the defocusing distance determination approaches based on sharpness metric, energy concentration criterion, amplitude modulus analysis, sparsity measurement, and different illumination modulations based analysis. Once the defocusing distance was identified with the above approaches, automatic focusing of moving samples could be realized, providing a powerful means for real-time observation of dynamic objects. Moreover, the applications of DHM auto-focusing technology in cell imaging, 3D particle tracking, etc were introduced as well.