In this study, we report the determination of dopamine (DA) and acetaminophen (AC) using activated graphene-Nafion modified glassy carbon electrode (AG-NA/GCE). The AG was prepared by simple and facile method of thermal activation of graphene oxide (GO) with potassium hydroxide (KOH) to enhance the pore volume and specific surface area. The surface morphology and pore structure of AG were characterized by field-emission scanning electron microscope, transmission electron microscopy, Raman spectroscopy and nitrogen isothermal adsorption -desorption technique. The electrochemical characterization of AG-NA/GCE was examined with cyclic voltammetry analysis of potassium ferricyanide. The electroanalytical performance of AG-NA/GCE toward the oxidation of DA and AC was analyzed via cyclic voltammetry and differential pulse voltammetry. The result showed excellent selectivity and sensitivity toward analysis of DA and AC with low detection limit values of 0.33 and 0.031 mu M (S/N = 3) for DA and AC, respectively. As for the examination and evaluation of electrochemical activities in real samples, the determination of DA and AC was examined with AG-NA/GCE in diluted human urine samples.