Abstract: As a micro-and nano-scale mechanical tool, Atomic Force Microscopy (AFM) is increasingly used in the experimental study of the biomechanics and promotes the further development of this interdisciplinary field. Using a variety of operation modes and modified probes, the AFM can carry out mechanical measurements on living matter at multiple scales, from subcellular structures to living cells and tissues. It can be used to study variations of the mechanical properties during different living processes, such as aging and cancerization. In this article, we will review the working principle of AFM, its experimental implementations in biomechanical measurements, and the applications of the AFM in the study of the mechanical properties of the whole cell and local variations, liquid-liquid phase-separated droplets, and epithelial cysts. We also analyze the effects of complex fluids and micro-and nano-scale flows on the AFM measurements, and make an outlook on the development of this field.