Taking into account the near-field coupling interaction, non-close-packed two-dimensional nanoparticle (NP) assemblies with centimeter-scale and tunable interparticle gap (d) have attracted considerable attention due to their remarkable physicochemical properties, which show a wide range of potential applications, e.g., surface-enhanced Raman spectroscopy (SERS) active substrates. In the present work, we demonstrate that an Au NP superlattice monolayer (SM) with tunable d, playing a critical role in governing SERS activity, can be created via a rapid liquid–liquid interfacial assembly strategy. We show that the enhancement factor (EF) of SERS has an approximate 1/d^2.4 dependence on the gap of Au NP SM assemblies. This work provides a platform for the rational design of plasmon-enhanced spectroscopy active substrates for theoretical studies and for various applications, including SERS-active substrates, photoelectronic devices, biosensors and others.