Progesterone is a precursor of many steroidal hormones and associated with the stabilization and control of hormonal levels in the reproductive cycle. Herein, the use of reduced graphene oxide (rGO), 5-Amino-2-mercaptobenzimidazole (AMBI) and gold nanoparticles (AuNPs) on Screen-Printing carbon electrode (SPCE) electrode as a modified electrode of electrochemical sensors for voltammetric determination of progesterone (P4) is described for the first time. Characterization was performed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Raman spectra. The properties of the modified electrode were examined by cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Under operating voltage of −1.64 V, the determination is performed by square wave voltammetry. Figures of merits such as linear dynamic response for P4 concentration ranging from 0.9 × 10−9 to 27 × 10−6 mol L−1, limit of detection of 0.28 × 10−9 mol L−1. For the first time, AuNP/AMBI/rGO electrode has been constructed and applied for high-performance P4 sensing, with potential for development in further electrochemical sensing applications.