We herein report the real-time observation of G-quadruplex formation by monitoring the G-quadruplex-induced global change of two duplexes incorporated in a DNA nanoscaffold. The introduced G-rich strands formed an interstrand (3 + 1) G-quadruplex structure in the presence of K+, and the formed four-stranded structure was disrupted by removal of K. These conformational changes were visualized in a nanoscaffold in real-time with fast-scanning atomic force microscopy.