The present work describes the electrochemical activity of dodecanethiol-capped gold nanoparticles (AuNPs) obtained by modified biphasic method. The variation of the stoichiometric ratio between [AuCl₄⁻] and dodecanethiol has yielded nanostructures with different sizes and distinct electrochemical activities. Using molar ratios Au:dodecanethiol (Au:Thiol) of 1:1, 2:1 and 4:1, gold nanoparticles were obtained with average diameter of 2.3 ± 0.7, 3.1 ± 0.5 and 4.3 ± 0.7 nm, respectively. Cyclic voltammetry (CV) experiments using glassy carbon electrode modified with such gold nanoparticles were performed and the influence of AuNPs amount, pH, scan rate sweep and current stability on the voltammetric response has been analysed. The system which has presented best electroactivity to oxide formation was evaluated as sensor for Diltiazem (DTZ) determination. Voltammetric response was observed in presence of DTZ attributed to electrochemical–chemical (EC’) mechanism between the analyte and the electrode surface. The cathodic peak currents obtained from Linear Sweep Voltammetry (LSV) decrease linearly with increasing DTZ concentrations, obtaining a linear profile for DTZ solutions ranging from 4.0 to 13.5 nmol L⁻¹ with limit of detection and limit of quantification of 0.5 and 1.7 nmol L⁻¹, respectively.