Polydopamine (PDA) has established itself as a promising grafting and coating material, particularly for functional group-deprived electrochemically active nanomaterials such as graphene, MXene, CNT, metal nanoparticles, and so on, and has proven its extensive applicability in the design and development of electrochemical biosensor devices. However, polynorepinephrine (PNE), a sister compound of PDA, having additional –OH groups and greater coating uniformity and biocompatibility, has never been studied in the field of biosensors. Herein, we investigated PNE as a coating material for reduced graphene oxide (RGO) and gold nanoparticles (Au) in order to build an electrochemical genosensor for Mycobacterium tuberculosis (MTB) detection. Biotin-Avidin chemistry was used to covalently immobilize probe DNA (ssDNA) specific to MTB to the nanocomposite surface on glassy carbon electrode (GCE) in order to construct biosensing electrodes. The formation of RGO/PNE and RGO/PNE/Au nanocomposite as well as the immobilization of ssDNA onto the bioelectrodes are both corroborated by UV–Visible, Raman, and XRD studies with FE-SEM and HR-TEM analysis. The electrochemical studies performed using cyclic voltammetry (CV) and linear sweep voltammetry (LSV) showed the significant enhancement in charge transfer kinetics of RGO/PNE/GCE and RGO/PNE/Au/GCE electrode compared to GO/GCE electrode. The biosensing investigations performed using ssDNA/avidin/RGO/PNE/Au/GCE bioelectrode showed high sensitivity (2.3 × 10−3 mA μM−1), low detection limit (0.1 × 10−7 μM), broad detection range (0.1 × 10−2 to 0.1 × 10−7 μM) with good selectivity and low response time (5 s) of the developed sensor. In comparison to the analogous RGO/PDA/Au material system, RGO/PNE/Au demonstrated increased enzyme loading, improved electrochemical responsiveness, and superior biosensing performance.