This work reports the synthesis of nitrogen and sulfur co-doped carbon dots (NS-CDs) via a one-step facile microwave-assisted pyrolysis using citric acid and thiourea as the carbon precursor and dopant, respectively. The resultant NS-CDs were found to be uniform in size and can be dispersed in aqueous solution. With an average size of 4.73 ± 0.65 nm, the NS-CDs exhibit excitation-dependent and pH-dependent photoluminescence properties in the visible range. Moreover, the NS-CDs also possessed a relatively high photoluminescence quantum yield of 28.9% and strong photostability in a high ionic strength environment. The NS-CDs were highly selective for Fe 3+ detection in a water sample, achieving a limit of detection (LOD) of 0.16 μM. On top of this, the NS-CDs also demonstrated high detection sensitivity with a LOD of 0.17 μM and improved selectivity to Fe 3+ in an acidic environment (pH 2). Specifically, the interference effect from Cu 2+ , Co 2+ , and Ag + was lowered by 15%, 18%, and 64% respectively, highlighting an additional merit for the as-synthesized NS-CDs. This concept of pH tuning can be implemented to exploit the detection capability in both neutral and acidic environments to create specific analytical responses correlating to a specific targeted metal ion. Finally, we also demonstrated the versatility of the NS-CDs by employing them as a low-toxicity fluorescent label and for semi-quantitative Fe 3+ sensing in cancer cells.