A facile electrochemical sensor based on carbon felt electrode (CFE) modified with gold nanoparticles decorated copper based metal organic framework (AuNPs@Cu-MOF) was achieved for the electrochemical sensing of L-Cysteine (L-Cys). For this purpose, AuNPs@Cu-MOF was synthesized and characterized. The electrochemical behaviors of L-Cys at plain and modified CFEs were investigated via cyclic voltammetry (CV). According CV results, AuNPs@Cu-MOF structure showed a catalytic effect on the oxidation of L-Cys as well as increasing the active electrode surface area by 206% compared to bare CFE. In addition, the pH effect on electrochemical determination of L-Cys at AuNPs@Cu-MOF/CFE was widely examined, and it was determined that the best oxidation peak current of L-Cys was obtained in pH 5 acetate buffer. Moreover, a linear detection range of 30–400 µM for L-Cys with a limit of detection value of 2.21 µM (n = 3) was achieved with the proposed electrochemical sensor. The developed L-Cys sensor was also applied for L-Cys detection in various milk samples and acceptable recovery values were obtained ranging from 100.05 to 108.45%.