To design a probe with “turn-on” sensing, nitrogen and sulfur co-doped carbon quantum dots (N, S-CQDs) were prepared and screened against some metallic cations to first induce “turn-off” fluorescence. The ferric iron (Fe3+) was shown to be the most responsive and effective in the fluorescence quenching of the N, S-CQDs based on a proposed photo-induced electron transfer mechanism. In addition, the fluorescence of N, S-CQDs-Fe3+ system was well recovered using N-acetyl-l-cysteine (NAC) (turn-on) due to a redox reaction, suggesting that the N, S-CQDs-Fe3+ system acts as a highly sensitive and selective sensor for the determination of N-acetyl-l-cysteine with a low limit of detection equal to 65.0 nmol/L and wide linear ranges from 0.67 to 25.56 and 25.56 to 193.55 μmol/L. The “turn-off/on” fluorescence method was successfully employed to monitor N-acetyl-l-cysteine in pharmaceutical products and human urine samples with a recovery range from 99.2 to 101.3%. In addition, the fluorescence switch properties of the nitrogen and sulfur co-doped carbon quantum dots were also investigated by alternate additions of Fe3+ and N-acetyl-l-cysteine.