The development of three-dimensional atomic force microscopy (3D-AFM) enabled the direct visualization of 3D hydration structures at solid-liquid interfaces with subnanometer resolution. Such imaging is possible because the hydration structure, once disorganized by the tip scan, can recover its original state through self-organization. Based on the same concept, the interior of any 3D self-organizing systems (3D-SOSs) may be visualized by 3D-AFM. To pursue this possibility, we have explored 3D-AFM imaging of various 3D-SOSs in interface sciences, life sciences and electrochemistry. Here, we review our recent progress in such 3D-AFM studies on 3D-SOSs, including hydration structures on cellulose nanocrystals, adsorption structures of anti-freezing surfactants on sapphire (0001) surfaces, intra-cellular components inside living cells, and charges accumulated inside an electric double layer. These examples demonstrate the effectiveness of 3D-AFM for understanding the nanoscale structures, properties and functions of various 3D-SOSs.