Noble metal nanoparticles (NPs) with unique plasmonic properties are currently the focus for high-performance surface-enhanced Raman scattering (SERS) substrates. However, how to effectively combine metal NPs with two-dimensional materials to further improve SERS detection sensitivity is still a great challenge. Herein, we successfully synthesize AuAg alloy NPs (ANPs) that integrate the desired plasmonic performance of Ag and the good stability of Au. The plasmonic properties of the AuAg ANPs can be easily tuned by changing the molar ratio of Au and Ag. In addition, we also develop a three-dimensional AuAg ANPs/graphene (G)/AuAg ANPs sandwiched hybrid nanostructure (SHNS) substrate by a reproducible interfacial self-assembly technique. The structure consists of two layers of AuAg ANPs film arrays using monolayer graphene as a nanospacer. The SHNS substrate with sub-nanometer gaps exhibits much higher SERS sensitivity and uniformity for the detection of Rh6G molecules, which is mainly due to the high-density “hot spots” in the nanogaps including between AuAg ANPs and AuAg ANPs in the same layer as well as in the monolayer graphene sub-nanospacer, the synergistic effect between Au and Ag species, and the additional chemical enhancement from graphene. The current studied strategy can be exploited for investigating SERS enhancement mechanisms and developing applications of SHNS substrates with graphene in biological sensing, biodetection, and catalysis.