We report on the synthesis of manganese oxide doped CDs (MnO-CDs) by a hydrothermal strategy using manganese (III) acetylacetonate (Mn(III) (CHO)) as the only raw materials. The MnO-CDs exhibit water solubility, favorable biocompatibility, low cytotoxicity, and show blue fluorescence with excitation/emission maxima at 326/442 nm with a quantum yield of 11.3%, allowing efficient cellular imaging. The MnO-CDs have a reversible temperature-sensitive fluorescent property in vitro within 10-60 °C, which can also be used as a sensitive thermometer in living cells. By a scratch assay, the MnO-CDs can restrain the migration of HepG2 cancer cells, which make the MnO-CDs be attractive candidates for liver cancer adjuvant treatment. Besides, the fluorescence of the MnO-CDs is quenched in the presence of Fe due to the formation of a nonfluorescent MnO-CDs-Fe complex between oxygen-containing groups on the surface of MnO-CDs and Fe, and the quenched fluorescence of MnO-CDs can be turn-on by dissociation of MnO-CDs-Fe complexes by biothiols including L-cysteine, homocysteine and glutathione. Therefore, the Fe and biothiols can be sequentially detected with high reliability and accuracy via exploiting the on-off-on nanosensor at room temperature, respectively. Further application to detection biothiols in human serum indicated that the probe was practicality and feasibility in medical field.