Using triiron tetroxide/magnetite particles with particle diameters of 5 nm and 30 nm, the structural color occurrence behavior due to multi‐particle layer formation was compared. The −OH‐terminated groups on the surface of the outermost layer of magnetite were bound with long‐chain fatty acids to obtain amphiphilic organo‐modified magnetite. Organo‐modified magnetite was spread as a single‐particle layer on the water surface and layered in multiple layers on a solid substrate. Organo‐modified magnetite multilayers layered to a thickness close to the wavelength of visible light exhibited a clear structural color, regardless of the particle size. However, in the case of fine particles with a diameter of 5 nm, the structural color did not appear when the in‐plane packing density was low, and the density of the two‐dimensional plane played an important role. In comparison, in the same production process and particle type, a clearer structural color was expressed with a diameter of 30 nm, and individual differences in physical properties were also confirmed when other nanoparticle types were used.