A novel nanobiosensor was constructed with graphene oxide (GO) sheets coupled to pear extract-based green-synthesised silver nanoparticles (Ag-NPs) to which cytochrome P450-2D6 (CYP2D6) enzyme was attached. The biosensor was applied in the electrochemical detection of the tuberculosis (TB) treatment drugs, ethambutol (EMB) and pyrazinamide (PZA). The surface morphology of the green-synthesised nanocomposites was studied by performing High-Resolution Transmission Electron Microscopy (HR-TEM) and High-Resolution Scanning Electron Microscopy (HR-SEM). Fourier Transform Infrared Spectroscopy (FTIR) and Raman Spectroscopy were used for structural analysis, while Ultraviolet Visible (UV-Vis) Spectroscopy was used in the optical characterisation of the nanocomposite material. Electrochemical studies on glassy carbon electrode (GCE), which were done by Cyclic Voltammetry (CV), showed that the GO|Ag-NPs||GCE electrode was highly conductive, and thereby indicating its suitability as a platform for nanobiosensor development. The non-toxic and low-cost green GO|Ag-NPs|CYP2D6||GCE nanobiosensor was used to determine EMB and PZA. The very low limit of detection (LOD) values of the biosensor for EMB (0.2962 × 10−2 nM, S/N = 3) and PZA (0.897 × 10−2 nM, S/N = 3) demonstrate that the green nanobiosensor is more sensitive than other biosensors reported for EMB and PZA.