Herein, we report a facial approach for the self-assembly of Au NP at the water–oil interface into a large-scale well-ordered, uniform-sized monolayer film with precise and tunable size of interparticle spacings and easily transferable properties. As a demonstration, a series of oil-soluble metallacycles containing disulfide end groups and water-soluble Au NPs with different size are employed. Supramolecular metallacycle-assisted interfacial self-assembly can be implemented by S-Au and electrostatic interaction at the water-dichloromethane interface to obtain Au NP monolayer films. The interparticle spacing of such monolayer films can be well-tuned by simply changing the size of the metallacycles. More importantly, the as-prepared monolayer film showed amphiphilic nature and could be easily transferred to a solid substrate for use as a suitable SERS substrate. It is highly anticipated that this work will provide a facile and general approach to realize control over self-assembly of Au NPs into well-defined nanostructures and pave a way to explore new nanomaterials with numerous plasmon-based applications.