HypothesisSelf-assembled nanostructures with highly ordered and diversified patterns can be obtained by adding additives that directionally control the interparticle interactions. However, due to the complex non-covalent weak interactions in the self-assembly process, the active mechanism of additives is not fully understood, resulting in the limitation of obtaining the nano-superstructures. The introduction of rhodamine 6G (R6G) enables gold nanorods (GNRs) self-assembled into a counterintuitive tetragonal superlattice, during which the exploration of the influence of R6G molecular interactions on the GNRs self-assembly is of importance. ExperimentsWe present the detailed investigations of spacial configuration, binding modes, and aggregated degree of R6G molecule on formation of the tetragonal GNRs superlattices by combining the experimental and simulated results. FindingsBy analyzing the peak position and peak intensity in the fluorescent spectra of assembled samples and pure R6G samples, H-dimer is verified as the main cause for inducing the tetragonal superstructures. Molecular dynamics simulations reveal that 2–3 H-dimers adsorbed obliquely in a zigzag chain manner on the surface of GNRs is the most stable state of the self-assembly. This work would contribute to a deeper understanding of the complex colloidal nanoparticle self-assemblies and push forward the development of the bottom-up nanoscale superstructures.