A selective electrochemical method is fabricated via layer-by-layer (LBL) method using both positively and negatively charged multi walled carbon nanotubes (MWCNTs) on poly (diallyldimetheylammonium chloride) (PDDA)/poly styrene sulfonate (PSS) modified graphite electrode, for the determination of acetaminophen (ACT) in the presence of dopamine (DA) and high concentration of ascorbic acid (AA). The modified electrode was characterized by cyclic voltammetry (CV) electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM) and scanning electron microscopy (SEM). Experimental conditions such as pH, accumulation potential and time, effect of potential sweep rates and interferents were studied. In CV well defined peaks for AA, ACT and DA are obtained at 24, 186 and 374 mV, respectively. The separations of peaks were 210, 188 and 398 mV between AA and DA, DA and ACT and AA and ACT, respectively. The diffusion coefficient was calculated by chronocoulometric. Chronoamperometric studies showed the linear relationship between oxidation peak current and concentration of ACT in the range 25–400 µM (R = 0.9991). The detection limit was 5 × 10⁻⁷ mol/L. The proposed method gave satisfactory results in the determination of ACT in pharmaceutical and human serum samples.