Highly red luminescent and water-soluble Ag₂S nanoclusters (NCs) are synthesized using glutathione as a stabilizing and capping agent. The as-prepared NCs have been characterized in terms of photoluminescence excitation spectrum, photoluminescence spectrum, transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. The NCs are shown to be viable fluorescent probes for the determination of Cu²⁺ ions due to aggregation-induced quenching of fluorescence. Furthermore, the nanosensor exhibits high sensitivity to Cu²⁺ ions with a detection limit of 2.5 nmol L⁻¹ and has been demonstrated for determination of Cu²⁺ ions in real water samples including tap water, mineral water, and Taihu lake water. In addition, the as-prepared NCs could be used as versatile nanothermometry devices in cellular and in in vivo temperature sensing based on obvious temperature dependence on the logarithm of fluorescence emission intensity, which changes obviously over the physiological temperature range (6–50 °C).