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 μM 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.