Due to the unique advantages (e.g., nanometer spatial resolution, picoNewton force sensitivity, label-free, can work in aqueous conditions), atomic force microscopy(AFM) has become an important instrument in cell biology. AFM can not only visualize the ultra-microstructures on the surface of living cells, but also can quantify the cellular mechanical properties (such as Young's modulus) by indenting technique, opening the doors to in situ explore the dynamical physiological activities and mechanical behaviors of single living cells at the nanoscale. In the past decades, researchers have carried out extensive investigations in imaging the cellular ultra-microstructures and measuring the cellular mechanical properties using AFM, yielding novel insights into our understanding of cellular physiological activities and providing a new idea to solve the related issues in the field of biomedicine. The AFM's own performances have also been steadily improved, which further promote its applications in biology. In this paper, based on our own research in investigating the killing effects of targeted cancer drugs at the nanoscale using AFM, the principle of AFM imaging and measuring the cellular mechanical properties was presented, the progress in visualizing the cellular ultra-microstructures and quantifying the cellular mechanical properties using AFM was summarized, the challenges facing AFM single-cell assay and its future directions were discussed.