A sensitive electrochemical sensor determination of dopamine (DA) has been developed using the polymerized L-tryptophan (p-L-Trp) and graphene (GN) composite as the electrode. The sensor electrode is fabricated by electrochemical polymerization of L-Trp on the GN-modified glassy carbon electrode. The composite sensor electrode shows an excellent electrochemical catalytic activity towards DA oxidation. The oxidation current is recorded by differential pulse voltammetry. The peak oxidation current is in logarithmically proportion to the DA concentration in a range from 0.2 to 100 μM with a detection limit of 0.06 μM. The detection selectivity is also carried out in the presence of ascorbic acid with a high concentration. The sensor is successfully applied for detecting DA in dopamine hydrochloride injection in real samples. The success of the sensor in DA detection is ascribed to a synergetic effect and π–π stacking interaction of GN and p-L-Trp.