This work presents the development of a sensor based on reduced graphene oxide (rGO) modified with silver nanoparticles (AgNPs) for the oxidation of estriol hormone. The morphology and electrochemical behavior of the rGO-AgNPs composite was characterized by scanning electron microscopy and cyclic voltammetry, demonstrating that the AgNPs were incorporated into rGO. The transfer of an electron from the estriol molecule for the working electrode at +0.45 V, and the consequent loss of an H+ ion for the buffer solution was confirmed combining electrochemical experiments and molecular modelling techniques. The glassy carbon (GC) electrode modified with the rGO/AgNPs composite was optimized for the determination of estriol using differential pulse voltammetry (DPV), achieving detection limit of 21.0 nmol L-1 for estriol hormone. Reproducibility and repeatability values of 1.5% and 1.3%, respectively, were obtained compared to the conventional procedure. The proposed GC/rGO-AgNPs electrochemical device was successfully applied to the determination of estriol in tap water and synthetic urine samples.