Temperature measurement in biology and medical diagnostics, along with sensitive temperature probing in living cells, is of great importance; however, it still faces significant challenges. Metal nanoclusters （NCs） with attractive luminescent properties may be promising candidates to overcome such challenges. Here, a novel one-step synthetic method is presented to prepare highly fluorescent copper NCs （CuNCs） in ambient conditions by using glutathione （GSH） as both the reducing agent and the protective layer preventing the aggregation of the as-formed NCs. The resultant CuNCs, with an average diameter of 2.3 nm, contain 1-3 atoms and exhibit red fluorescence （A~m = 610 nm） with high quantum yields （QYs, up to 5.0%）. Interestingly, the fluorescence signal of the CuNCs is reversibly responsive to the environmental temperature in the range of 15-80 ℃. Furthermore, as the CuNCs exhibit good biocompatibility, they can pervade the MC3T3-E1 cells and enable measurements over the physiological temperature range of 15-45 ℃ with the use of the confocal fluorescence imaging method. In view of the facile synthesis method and attractive fluorescence properties, the as-prepared CuNCs may be used as photoluminescence thermometers and biosensors.