© 2019 John Wiley & Sons, Ltd. Surface enhanced Raman spectroscopy (SERS) holds great promise in biosensing because of its single-molecule, label-free sensitivity. We describe here the use of a graphene–gold hybrid plasmonic platform that enables quantitative SERS measurement. Quantification is enabled by normalizing analyte peak intensities to that of the graphene G peak. We show that two complementary quantification modes are intrinsic features of the platform and that through their combined use, the platform enables accurate determination of analyte concentration over a concentration range spanning seven orders of magnitude. We demonstrate, using a biologically relevant test analyte, the amyloid β-protein (Aβ), a seminal pathologic agent of Alzheimer's disease, that linear relationships exist between (a) peak intensity and concentration at a single plasmonic hot spot smaller than 100 nm and (b) frequency of hot spots with observable protein signals, that is, the colocation of an Aβ protein and a hot spot. We demonstrate the detection of Aβ at a concentration as low as 10−18 M after a single 20 μl aliquot of the analyte onto the hybrid platform. This detection sensitivity can be improved further through multiple applications of analyte to the platform and by rastering the laser beam with smaller step sizes.