A unique nanocomposite was fabricated using negatively charged manganese dioxide nanoparticles, poly (3,4-ethylenedioxythiophene) and reduced graphene oxide (MnO/PEDOT/rGO). The nanocomposite was deposited on a glassy carbon electrode (GCE) functionalized with amino groups. The modified GCE was used to electrochemically detect dopamine (DA). The surface morphology, charge effect and electrochemical behaviours of the modified GCE were characterized by scanning electron microscopy, energy dispersive X-ray analysis (EDX), cyclic voltammetry and electrochemical impedance spectroscopy, respectively. The MnO/PEDOT/rGO/GCE exhibited excellent performance towards DA sensing with a linear range between 0.05 and 135 µM with a lowest detection limit of 30 nM (S/N = 3). Selectivity towards DA was high in the presence of high concentrations of the typical interferences ascorbic acid and uric acid. The stability and reproducibility of the electrode were good. The sensor accurately determined DA in human serum. The synergic effect of the multiple components of the fabricated nanocomposite were critical to the good DA sensing performance. rGO provided a conductive backbone, PEDOT directed the uniform growth of MnO and adsorbed DA via pi-pi and electrostatic interaction, while the negatively charged MnO provided adsorption and catalytic sites for protonated DA. This work produced a promising biosensor that sensitively and selectively detected DA.