Sulfur-doped carbon dots (S-CDs) with a quantum yield (QY) of 5.5% and nitrogen, sulfur co-doped carbon dots (N,S-CDs) with a QY of 54.4% were synthesized, respectively, via the same hydrothermal route using α-lipoic acid as the carbon source. The obtained S-CDs and N,S-CDs had similar sizes but different optical features. The QY of N,S-CDs was gradually enhanced when extending the reaction time to increase the nitrogen content. After careful characterization of these CDs, the doped nitrogen element was believed to be in the form of C=N and C-N bonds which enhanced the fluorescence efficiency significantly. Meanwhile, the co-doped sulfur element was found to be synergistic for nitrogen doping in N,S-CDs. The optimal N,S-CDs were successfully employed as good multicolor cell imaging probes due to their fine dispersion in water, excitation-dependent emission, excellent fluorescence stability and low toxicity. Besides, such N,S-CDs showed a wide detection range and excellent accuracy as fluorescent sensors for Fe(3+) ions.