The nitrogen and sulfur co-doped carbon dots (NSCDs) were synthesized using cysteine as precursor by hydrothermal approach, which showed good aqueous solubility, high fluorescent quantum yield (QY = 38.7%) and good photostability. The experiments with cells demonstrated that NSCDs possessed excellent biocompatibility and their cytotoxicity was low (cell viability >= 90%) in both normal and cancer cell lines. Furthermore, NSCDs as fluorescent probes could be used for the imaging of cells, which showed a good membrane permeability and strong resistance to photobleaching. In addition, the cellular uptake of NSCDs presented a time-dependent saturation. The imaging applicability of NSCDs in different cancer cells (MCF-7, A375, Hela and U251 cells) has also been demonstrated. Therefore, NSCDs are considered as promising candidates for the design of low cytotoxicity bioimaging agents and a cellular location system based on fluorescence measurements.