The identification of cysteine enantiomers is of great significance in the biopharmaceutical industry and medical diagnostics. Herein, we develop an electrochemical sensor to discriminate cysteine (Cys) enantiomers based on the integration of a copper metal-organic framework (Cu-MOF) with an ionic liquid. Because the combine energy of D-cysteine (D-Cys) with Cu-MOF (-9.905 eV) is lower than that of L-cysteine (L-Cys) with Cu-MOF (-9.694 eV), the decrease in the peak current of the Cu-MOF/GCE induced by D-Cys is slightly higher than that induced by L-Cys in the absence of an ionic liquid. In contrast, the combine energy of L-Cys with an ionic liquid (-1.084 eV) is lower than that of D-Cys with an ionic liquid (-1.052 eV), and the ionic liquid is easier to cross-link with L-Cys than with D-Cys. When an ionic liquid is present, the decrease in the peak current of the Cu-MOF/GCE induced by D-Cys is much higher than that induced by L-Cys. Consequently, this electrochemical sensor can efficiently discriminate D-Cys from L-Cys, and it can sensitively detect D-Cys with a detection limit of 0.38 nM. Moreover, this electrochemical sensor exhibits good selectivity, and it can accurately measure the spiked D-Cys in human serum with a recovery ratio of 100.2-102.6%, with wide applications in biomedical research and drug discovery.