Cysteine is one of the body’s essential amino acids to build proteins. For the early diagnosis of a number of diseases and biological issues, L-cysteine (L-Cys) is essential. Our study presents an electrochemical sensor that detects L-cysteine by immobilizing the horseradish peroxidase (HRP) enzyme on a reduced graphene oxide (GCE) modified glassy carbon electrode. The morphologies and chemical compositions of synthesized materials were examined using Fourier transform infrared spectroscopy (FTIR) and field-emission scanning electron microscopy (FESEM). The modified electrode’s electrochemical behavior was investigated using cyclic voltammetry (CV). Cyclic voltammetry demonstrated HRP/rGO/GCE has better electrocatalytic activity than bare GCE in the oxidation of L-cysteine oxidation in a solution of acetate buffer. The electrochemical sensor had a broad linear range of 0 µM to 1 mM, a 0.32 µM detection limit, and a sensitivity of 6.08 μA μM⁻¹ cm⁻². The developed sensor was successfully used for the L-cysteine detection in a real blood sample with good results.