In the present study, multiwalled carbon nanotubes(MWCNTs), gold nanoparticles(AuN Ps), and glutathione(GSH) were used to fabricate multilayer nanoscale thin films. The composite thin films were fabricated by layer-by-layer technique as the films were constructed by the alternate deposition of cationic and anionic polyelectrolytes. The MWCNTs were modified via a noncovalent surface modification method using poly(diallydimethylammonium chloride) to form a cationic polyelectrolyte. An anionic polyelectrolyte was prepared by the chemical reduction of HAuCl_4 using sodium citrate as both the stabilizing and reducing agent to form anionic AuN Ps. GSH was used as an electrocatalyst toward the electro-oxidation of dopamine. The constructed composite electrode exhibits excellent electrocatalytic activity toward dopamine with a short response time and a wide linear range from 1 to 100 mmol/L. The limits of detection and quantitation of dopamine are(0.316 ± 0.081) mmol/L and(1.054 ± 0.081) mmol/L, respectively. The method is satisfactorily applied for the determination of dopamine in plasma and urine samples to obtain the recovery in the range from 97.90% to 105.00%. In the present study, multiwalled carbon nanotubes (MWCNTs), gold nanoparticles (AuN Ps), and glutathione (GSH) were used to fabricate multilayer nanoscale thin films. The composite thin films were fabricated by layer-by-layer technique as the films were constructed by the alternate deposition of cationic and anionic polyelectrolytes. The MWCNTs were modified via a noncovalent surface modification method using poly (diallydimethylammonium chloride) to form a cationic polyelectrolyte. An anionic polyelectrolyte was prepared by the chemical reduction of HAuCl_4 using sodium citrate as both the stabilizing and reducing agent to form anionic AuN Ps. GSH was used as an electrocatalyst toward the electro-oxidation of dopamine. The constructed composite electrode exhibits excellent electrocatalytic activity toward dopamine with a short response time and a wide linear range from 1 to 100 mmol / L. The limits of detection and quantitation of dopamine are (0.316 ± 0.081) mmol / L and (1.054 ± 0.081) mmo l / L, respectively. The method is satisfactorily applied for the determination of dopamine in plasma and urine samples to obtain the recovery in the range from 97.90% to 105.00%.