A reliable method to prepare a surface-enhanced Raman scattering (SERS) active substrate is developed herein, by electrodeposition of gold nanoparticles (Au NPs) on defect-engineered, large area CVD graphene (GR). A plasma treatment strategy has been used in order to engineer the structural defects on the basal plane of large area single-layer graphene. This defect-engineered Au functionalised GR, offers reproducible SERS signals over the large area GR surface. The Raman data, along with X-ray photoelectron spectroscopy and analysis of the water contact angle are used to rationalise the functionalization of the graphene layer. We find that Au NPs functionalisation of the “defect-engineered” graphene substrates permits detection of concentrations as low as 10−16 M for the probe molecule Rhodamine B (RhB), which offers an outstanding molecular sensing ability. Interestingly, a Raman signal enhancement of up to∼ 108 was achieved. Moreover, we observed that GR effectively quenches the fluorescence background from the Au NPs and molecules due to the strong resonance energy transfer between Au NPs and GR. The results presented offer significant direction for the design and fabrication of ultra-sensitive SERS platforms, and also open up possibilities for novel applications of defect engineered graphene in biosensors, catalysis, and optoelectronic devices.