The advent of atomic force microscopy (AFM) provides a powerful tool for imaging individual DNA molecules. Chemotherapy drugs are often related to DNAs. Though many specific drug-DNA interactions have been observed by AFM, knowledge about the dynamic interactions between chemotherapy drugs and plasmid DNAs is still scarce. In this work, AFM was applied to investigate the nanoscale interactions between plasmid DNAs and two commercial chemotherapy drugs (methotrexate and cisplatin). Plasmid DNAs were immobilized on mica which was coated by silanes in advance. AFM imaging distinctly revealed the dynamic changes of single plasmid DNAs after the stimulation of methotrexate and cisplatin. Geometric features of plasmid DNAs were extracted from AFM images and the statistical results showed that the geometric features of plasmid DNAs changed significantly after the stimulation of drugs. This research provides a novel idea to study the actions of chemotherapy drugs against plasmid DNAs at the single-molecule level.