l-3,4-Dihydroxy-phenylalanine (l-DOPA) is the most effective drug for the treatment of Parkinson's disease, which plays a very important role in clinical and neurochemistry. However, how to achieve high-sensitivity recognition of l-DOPA still faces challenges. Here, a facile strategy is presented to construct nitrogen-doped chiral CuO/CoO nanofibers (N-CuO/CoO NFs) with nanozyme activity and electrochemiluminescence property, in which CuO/CoO NFs are used as the catalytic activity center and chiral cysteine (Cys) is used as the inducer of chiral recognition, for enantioselective catalysis and sensitive recognition of DOPA enantiomers. Notably, N doping not only enhances the enzyme-mimic activity of CuO/CoO NFs but also amplifies their electrochemiluminescence (ECL) signals in the presence of luminol. More importantly, in the presence of DOPA enantiomers, the d-cysteine (d-Cys)-modified N-CuO/CoO NFs exhibit different ECL performances; thus, d-Cys@N-CuO/CoO NFs could selectively distinguish and sensitively detect l-DOPA through ECL signals, and the detection limit is 0.29 nM for l-DOPA. In addition, it also showed good sensing performance for the determination of l-DOPA in fetal bovine serum. This is the first report on the detection of DOPA enantiomers based on an enhanced ECL strategy, providing a robust pathway for chiral discrimination and detection of chiral molecules.