In this study, a novel Au nanocluster (NC)-based fluorescent sensor has been designed for near-infrared (NIR) and turn-on sensing of glutathione (GSH) in both living cells and human blood samples. The large Stokes-shifted (140 nm) fluorescent Au NCs with NIR emission and long-wavelength excitation have been rapidly synthesized for 2 h by means of a microwave-assisted method in aqueous solution. The addition of Hg-II leads to an almost complete emission quenching (98%) of Au NCs because of the interaction of Hg-II and Au-I on the surface of Au NCs. After introducing GSH to the Au NC-Hg-II system, a more than 20 times fluorescent enhancement is obtained because of the preferable affinity of GSH with Hg-II. Under optimum conditions, the fluorescence recovery is linearly proportional to the concentration of GSH between 0.04 and 16.0 mu M and the detection limit is as low as 7.0 nM. This Au NC-based sensor with high sensitivity and low spectral interference has been proven to facilitate biosensing applications.