Among the approaches for DNA-based drug targeting, G-quadruplex-binding ligands are of particular interest because of high abundance of G-rich sequences in regions such as human chromosomal telomeres and promoters of several proto-oncogenes. Number of quadruplex-ligands has been reported, but their functions at single-molecule level have not been explored using direct and real-time methods. Here, we report on the direct observation of the formation of a four-stranded G-quadruplex induced by bisquinolinium pyridine dicarboxamide with a linker containing biotin at one end. We fabricated a DNA origami frame with incorporated duplex DNAs that contained 3-6 G–G mismatches in the middle. In the absence of ligand, the duplex DNAs of interest had no interaction, as visualized by their parallel-shape in high-speed atomic force microscopy (HS-AFM) image. Presence of ligand induced the formation of G-quadruplex structure, which was characterized by an X-shape. Addition of streptavidin to the ligand-induced quadruplex caused the protein to localize in the middle of X-shape, indicating that the ligand is bound to the quadruplex. A sequence of real-time images of the ligand-induced formation of a quadruplex and its reverse conformational switching by removing the ligand was captured by HS-AFM. Unprecedented intermediate-like states were recorded in our real-time analysis.