Trimetazidine hydrochloride (TMZ), well-known by its unique and interesting pharmacological properties, has been investigated by several electrochemical techniques using Poly(3,4-ethylenedioxythiophene) coated glassy carbon electrode (GCE). The preparation of the modified electrode was achieved by the electrodeposition of PEDOT in two different media and the obtained film has been characterized by scanning electron microscopy (SEM) and infrared spectroscopy (FTIR). The electrochemical analysis of TMZ was then studied by cyclic, differential pulse and square wave voltammetric methods in solutions of different pH values and results have shown that square wave voltammetry (SWV) is the most suitable one for the analytical characterization. The oxidation of TMZ was irreversible and was a process under diffusion control. Besides, Model compounds and molecules were also used in this study to describe the possible oxidation mechanism of TMZ. In this contribution, the main objective is the possible practical use of the proposed method, thus, the electrochemical detection of TMZ by SWV was carried out in biological samples, such as human serum and urine. Under the optimum condition in phosphate buffer at pH 3.0, linear calibration curves were obtained for all studied media (supporting electrolyte, urine and serum). Important parameters, namely, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were reported, and good sensitivity, reproducibility and repeatability were obtained. Furthermore, recovery values of almost 100% were found for pharmaceuticals and biological samples, suggesting that the sensor can be used in practical applications.