In this work, an electrochemical sensor is fabricated by decorating the surface of graphite electrode with NiO/graphene (NGMG) nanoparticles and employed for the detection of dopamine (DA), tyrosine (Tyr) and ascorbic acid (AA). The structure and morphology of prepared NiO nanoparticles are examined by XRD,SEM, FTIR and Raman techniques. The electrochemical properties have been investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperommety. The modified electrode is prepared by a simple drop casting method. The electrode shows good electro catalyticactivity towards oxidation of DA, Tyr and AA. It successfully separates the oxidation current signals of AA, DA and Tyr into clearly visible three distinct oxidation peaks compared to a single, overlapped oxidative peak on bare graphite electrode. The peak potential difference between AA-DA, DA-Tyr and AA-Tyr is 228 mV, 303 mV and 565 mV respectively in cyclic voltammetry (CV) studies and the corresponding peak potential separations are 243 mV, 318 mV and 561 mV respectively in differential pulse voltammetry (DPV). It is found that oxidation mechanism of DA, AA and Tyr on NGMG are different owing to a different type of interaction of the modified layer with the bio-analytes. The modified electrode, NGMG has high selectivity and sensitivity in addition to other factors like low cost, convenient and a hassle free electrochemical method for simultaneous determination of DA, AA and Tyr in their ternary mixture.