The sensitivity of graphene’s Raman characteristics to external perturbations can be exploited for sensing applications. To apply this principle, Raman spectroscopy of graphene in contact with a chemical/biological analyte is recorded to unravel a correlation between analyte’s physical/chemical/biological properties and electronic properties (Fermi energy and dopants level) of graphene. As an example, we demonstrate the principle and operation of glucose measurement using graphene’s Raman spectroscopy, utilizing both affinity and enzymatic detection methods. In the affinity detection, we monitor Raman spectrum of graphene in contact with a glucose containing sample to quantify the attachment of glucose onto 1-pyrene boronic acid (PBA) functionalized graphene. It is revealed that glucose covalent bond to PBA induces n-type doping in graphene and increases its Fermi level following the Hill–Langmuir equation. In the enzymatic detection, we show that graphene’s Fermi energy shifts due to the biocatalytic oxidation of glucose by immobilized glucose oxidase (GOx) enzymes. As a result, graphene becomes p-doped with increasing glucose concentration. We attribute this sensitivity to a re-distribution of ionic charges within the electric double layer (EDL) of graphene upon introducing the glucose molecules. This model explains both our affinity and enzymatic glucose detection experiments. Beyond glucose detection, our graphene Raman spectroscopy-based sensor (GRS) may extend its applications to a broader range of remote and non-invasive detection without the need to employ electronics such as field-effect transistors.