In this study, in situ sulfur-doped carbon nitride nanosheets (S-g-C3N4 NSs) are used as the signal readout for the sensitive and selective sensing of l-cysteine (l-Cys) in human serum samples based on the competitive coordination chemistry of Cu2+ between l-Cys and S-g-C3N4 NSs. S-g-C3N4 NSs are prepared by using trithiocyanuric acid as a precursor for the first time, which exhibits stronger electrogenerated chemiluminescence (ECL) intensity compared with pristine graphitic carbon nitride nanosheets (g-C3N4 NSs). The ECL signals of the S-g-C3N4 NSs can be quenched by Cu2+ and the subsequent presence of l-Cys can recover the ECL signals of the S-g-C3N4 NSs. These changes are ascribed to the higher coordination ability between Cu2+ and l-Cys than that between Cu2+ and the S-g-C3N4 NSs. On the basis of this, the concentration of l-Cys can be quantitatively determined. Under optimized conditions, the ECL intensity recovery shows a linear relationship with the l-Cys concentration range from 30 nM to 0.2 mM with a lower detection limit of 5 nM (S/N = 3). The proposed method is highly sensitive and selective and is thus particularly useful for fast and simple clinical diagnosis of diseases, such as Alzheimer's disease and cardiovascular disease.