In recent years, the possibility of nanobubbles at the solid–liquid interface has drawn wide attention in the scientific community and industry. Thus the search for evidences for the existence of nanobubbles became a scientific hotspot. To produce interfacial nanobubbles, a systematic experiment, called the temperature difference method, is carried out by replacing low temperature water (LTW) with high temperature water (HTW) at the highly-oriented pyrolytic graphite (HOPG)–water interface. When LTW (4 °C) is mixed with HTW (25–40 °C), nanobubbles are observed by atomic force microscopy (AFM), and their size, density and total volume per square micrometer are measured. Furthermore, pancake-like gas layers and the coexistence of nanobubbles on top of the pancake layers are also observed.