Deoxyribonucleic acid (DNA), the structural basis of genetics, plays a crucial role in the life of cells. The abnormal behaviors of DNA are closely related to the pathological changes of living organisms. The advent of atomic forcemicroscopy (AFM) provides an exciting instrument for biophysically probing the nanoscale dynamic activities of the individual native molecules, considerably complementing the results obtained by traditional biochemical population-averaged measurements. In the past decades, AFM has been widely used in DNA studies, significantly providing novel insights into molecular biology. In this paper, the methodologies and progresses of utilizing AFM to characterize the behaviors of individual DNA molecules from several facets (including DNA topography imaging, DNA mechanics measurements, DNA origami, and DNA nanorobotics) are reviewed, and the future directions are discussed.