The conformation and alignment of molecules in organic materials are important because they affect the materials' bulk physical properties. Because two-dimensional (2D) materials offer a simpler model of three-dimensional (3D) materials, the conformation and alignment of molecules in 2D assemblies have been investigated at the atomic scale by scanning tunnelling microscopy (STM). However, differences in the conformation and alignment of molecules between 2D and 3D assemblies have not been clarified. In this work, the conformation and alignment of a donor-acceptor-type molecule, 4-(3,3-dimethyl-2,3-dihydro-1H-indol-1-yl)benzonitrile (IBN), are studied in 2D and 3D assemblies. Thus, the 2D assembly of IBN on the Au(111) surface was investigated by STM and the 3D assembly of IBN in a single crystal was investigated by X-ray crystallography. Our survey revealed that the conformation of IBN is planar in both 2D and 3D assemblies because of the electron-delocalised structure resulting from the electron-donating and electron-accepting groups of IBN; thus, the values of the dipole moment of IBN in 2D and 3D assemblies are essentially the same. In both the 2D and 3D assemblies, IBN molecules align to cancel out the dipole moment even though the self-assembled structures differ. In the 2D assemblies, the orientation and self-assembled structure of IBN are changed by the surface density of IBN, and they are affected by the crystal orientation and superstructure of Au(111) because of the strong interaction between IBN and Au(111). In addition, scanning tunnelling spectroscopy revealed that the coordination structure is not included in the self-assembled structure of IBN on Au(111).