Carbon dots have unique advantages in biological applications owing to their excellent optical properties. However, the biosafety evaluation of carbon dots has limitations owing to cytotoxicity in vitro, and there is little pre-safety evaluation before in vivo and clinical applications. Whether the carbon dots are or not suitable for applications in vivo, evaluation analysis can be made based on hemolysis and changes in erythrocyte morphology. In this work, a green fluorescent N, S-doped carbon dots (N, S-CDs) were obtained by hydrothermal method, tobias acid, and m-phenylenediamine as precursors. N, S-CDs not only possessed excellent dispersibility, uniform particle size, high quantum yield (37.2％) and stable photoluminescence property but also retain their photostability and strong fluorescence intensity in the acid/alkaline solutions, different ionic strengths (NaCl) and under 365 nm UV illumination. Moreover, the N, S-CDs displayed low cytotoxicity and high cellular uptake efficiency in human umbilical vein endothelial cells (HUVEC) and excellent blood compatibility to the erythrocyte. It is foreseeable that N, S-CDs could be further studied as a promising biological imaging agent in vivo.