This study presents a novel electrochemical sensing technique for the quantitative analysis of glutathione levels, a crucial biomolecule linked to various medical conditions such as diabetes, Parkinson's disease, and cancer. The increasing demand for simple, rapid, and cost-effective assays in clinical diagnostics drives the need for innovative solutions. We synthesized the metal-organic framework (MOF) MIL-101(Fe) via a solvothermal method and characterized using Fourier-transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), and energy dispersive x-ray spectroscopy (EDX). Then, a modified carbon paste electrode using MIL-101 (Fe) MOF and ionic liquid (IL) for ferrocene (FC)-mediated electrochemical determination of glutathione was prepared (MIL-101 (Fe) MOF/FC/ILCPE). The electrochemical performance of the designed electrode was assessed through cyclic voltammetry (CV), linear sweep voltammetry (LSV), chronoamperometry, and differential pulse voltammetry (DPV). Based on the CV investigations, the as-prepared MIL-101 (Fe) MOF/FC/ILCPE demonstrated good electrocatalytic performance towards the oxidation of glutathione. The developed sensor demonstrated a broad linear dynamic range of 0.5–385.0 µM for glutathione detection. The limit of detection (LOD) was calculated as 0.15 µM. Practical applications were validated through the analysis of human urine and hemolyzed erythrocyte samples, showing exceptional effectiveness in glutathione detection. This electrochemical sensor exhibits significant potential for clinical applications in monitoring glutathione levels.