In this paper, AuNPs@Polyethyleneimine-derived carbon hollow spheres were synthesized by a versatile and facile method in three steps and successfully developed and validated as Amitriptyline sensor using cyclic voltammetry (CV), chronoamperometry (CA) and differential pulse voltammetry (DPV) methods. The characterization of the electrode surface has been carried out by means of scanning electron microscopy (SEM), transmission electron microscopy (TEM), x-ray diffraction (XRD), x-ray photo-electron spectrum (XPS), electrochemical impedance spectroscopy (EIS) and chronocoulometry (CC). The obtained negatively charged modified electrode was highly selective to Amitriptyline and it was shown a wide linear range from 0.1 to 700 μmol L⁻¹, with a lower detection limit of 0.034 μmol L⁻¹ (n=5, S/N=3), revealing the high-sensitivity properties. The modified electrode is used to achieve the real-time quantitative detection of AMT for biological applications, and satisfactory results are obtained. Due to the advantages of the sensor, its selectivity, sensitivity and stability, it will have a bright future in the field of medical diagnosis.