Graphene quantum dots (GQDs), derived from functionalized graphene precursors are graphene sheets a few nanometers in the lateral dimension having a several-layer thickness. They are zero-dimensional materials with quantum confinement and edge site effects. Intense research interest in GQDs is attributed to their unique physicochemical phenomena arising from the sp²-bonded carbon nanocore surrounded with edged plane functional moieties. In this work, GQDs are synthesized by both solvothermal and hydrothermal techniques, with the optimal size of 5 nm determined using high-resolution transmission electron microscopy, with additional UV-Vis absorption and fluorescence spectroscopy, revealing electronic band signatures in the blue-violet region. Their potential in fundamental (direct electron transfer) and applied (enzyme-based glucose biosensor) electrochemistry has been practically realized. Glucose oxidase (GO_x) was immobilized on glassy carbon (GC) electrodes modified with GQDs and functionalized graphene (graphene oxide and reduced form). The cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy are used for characterizing the direct electron transfer kinetics and electrocatalytical biosensing. The well-defined quasi-reversible redox peaks were observed under various electrochemical environment and conditions (pH, concentration, scan rate) to determine the diffusion coefficient (D) and first-order electron transfer rate (k_ET). The cyclic voltammetry curves showed homogeneous ion transport behavior for GQD and other graphene-based samples with D ranging between 8.45 × 10⁻⁹ m² s⁻¹ and 3 × 10⁻⁸ m² s⁻¹ following the order of GO < rGO < GQD < GQD (with FcMeOH as redox probe) < GO_x/rGO < GO_x/GO < HRP/GQDs < GO_x/GQDs. The developed GO_x-GQDs biosensor responds efficiently and linearly to the presence of glucose over concentrations ranging between 10 μM and 3 mM with a limit of detection of 1.35 μM and sensitivity of 0.00769 μA μM⁻¹·cm⁻² as compared with rGO (0.025 μA μM⁻¹ cm⁻², 4.16 μM) and GO (0.064 μA μM⁻¹ cm⁻², 4.82 μM) nanosheets. The relatively high performance and stability of GQDs is attributed to a sufficiently large surface-to-volume ratio, excellent biocompatibility, abundant hydrophilic edges, and a partially hydrophobic plane that favors GO_x adsorption on the electrode surface and versatile architectures to ensure rapid charge transfer and electron/ion conduction (<10 ms). We also carried out similar studies with other enzymatic protein biomolecules on electrode surfaces prepared from GQD precursors for electrochemical comparison, thus opening up potential sensing applications in medicine as well as bio-nanotechnology.