High-speed AFM (HS-AFM) is an advanced technique with numerous applications in biology, particularly in molecular biophysics. Developed as a time-lapse AFM technique for direct imaging fully hydrated biological molecules, HS-AFM is currently capable of visualizing the dynamics of biological molecules and their complexes at a video-data acquisition rate. Spatial resolution at the nanometer level is another important characteristic of HS-AFM. This review focuses on examples of primarily protein-DNA complexes to illustrate the high temporal and spatial resolution capabilities of HS-AFM that have resulted in novel models and/or the functional mechanisms of these biological systems.