Copper nanoclusters (Cu NCs) are generally formed by several to dozens of atoms. Because of wide range of raw materials and cheap prices, Cu NCs have attracted scientists’ special attention. However, Cu NCs tend to undergo oxidation easily. Thus, there is a dire need to develop a synthetic protocol for preparing fluorescent Cu NCs with high QY and better stability. Herein, we report a one-step method for preparing stable blue-green fluorescent copper nanoclusters using glutathione (GSH) as both a reducing agent and a stabilizing agent. High-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS) and electrospray ionization mass spectrometer (ESI-MS) were used to characterize the resulting Cu NCs. The as-prepared Cu NCs@GSH possess an ultrasmall size (2.3 ± 0.4 nm), blue-green fluorescence with decent quantum yield (6.2%) and good stability. MTT results clearly suggest that the Cu NCs@GSH are biocompatible. After incubated with EB-labeled HEK293T cells, the Cu NCs mainly accumulated in nuclei of the cells, suggesting that the as-prepared Cu NCs could potentially be used as the fluorescent probe for applications in cellular imaging.