Abstract: Without losing its high resolution, high-speed atomic force microscope (HS-AFM) represents a perfect combination of scanning speed and precision and allows real-time and in situ observation of the dynamic processes in a biological system at both the cellular and molecular levels. By combining the extremely high temporal resolution with the spatial resolution and coupling with other advanced technologies, HS-AFM shows promising prospects for applications in life sciences such as cell biology. In this review, we summarize the latest progress of HS-AFM in the field of cell biology, and discuss the impact of environmental factors on conformation dynamics of DNA, the binding processes between DNA and protein, the domain changes of membrane proteins, motility of myosin, and surface structure changes of living cells.