Herein, the assembly behavior of gold nanoparticles (AuNPs) at the oil/water interface is studied by surface-enhanced Raman scattering (SERS) spectroscopy. Two selected chemicals [1-dodecanethiol (DDT) and tetramethylammonium ion (TMA(+))] are applied to tune the surface properties of AuNPs and the corresponding assembly behaviors at the oil/water interface are thoroughly investigated. Various AuNPs films, namely sparse 2D film, perfect monolayer, and multilayers are obtained. The SERS spectra analyses show that the surface composition of AuNPs is strongly dependent on the chemical environment around AuNPs and results in different morphologies of AuNPs film at the oil/water interface. Accordingly, we propose a rational relationship between AuNPs assembly behavior at the oil/water interface and their surrounding chemical environment, and thus reveals the physical mechanism underlying the nanoparticle assembly.