In this study, a novel electrochemical assay for determining 17-β-estradiol (E2) was proposed. The approach involves modifying a glassy carbon electrode (GCE) with a nanocomposite consisting of α-Fe2O3 nanoparticles supported on carbon nanotubes (CNTs)—denoted as α-Fe2O3-CNT/GCE. The synthesis of the α-Fe2O3-CNT nanocomposite was achieved through a simple and cost-effective hydrothermal process. Morphological and chemical characterization were conducted using scanning electron microscopy (SEM), Raman spectroscopy, and energy-dispersive X-ray spectroscopy (EDX). The presence of the α-Fe2O3-CNT film on the GCE surface resulted in an enhanced electrochemical response to E2, preventing electrode surface fouling and mitigating the decrease in peak current intensity during E2 oxidation. These outcomes substantiate the rationale behind the GCE modification. After the optimization of experimental conditions, E2 was determined by the square wave voltammetry technique using 0.1 mol L−1 KCl solution (pH = 7.0) with 20% ethanol as a supporting electrolyte. A linear concentration range of 5.0–100.0 nmol L−1 and a low limit of detection of 4.4 nmol L−1 were obtained. The electroanalytical method using α-Fe2O3-CNT/GCE was applied for E2 determination in pharmaceutical, lake water, and synthetic urine samples. The obtained results were attested by recovery tests and by high-performance liquid chromatography as a comparative technique at a 95% confidence level. Thus, the developed electrochemical sensor is simple and fast to obtain, presents high accuracy, and is viable for determining E2 in routine analysis.