This paper reports a quantum dot (QD)-based "off-on" fluorescent biosensor specifically for the determination of glutathione (GSH) with high sensitivity. The biosensor was based on the following two properties. Firstly, the high fluorescence of N-acetyl-L-cysteine (NALC) capped CdTe QDs could be effectively quenched by Hg(2+) due to the binding of Hg(2+) to the NALC on the surface of the QDs and the electron transfer from the photoexcited NALC-capped CdTe QDs to Hg(2+). Secondly, in the presence of GSH, the fluorescence intensity of NALC-capped CdTe QDs was found to be efficiently recovered. Under some optimized conditions, the relatively restored fluorescence intensity was proportional to the concentration of GSH in the range of 4-64 μg mL(-1), with a correlation coefficient of 0.9980 and a limit of detection of 2.49 ng mL(-1). In addition, the established method shows a high selectivity for some amino acids except cysteine. Moreover, to further investigate its performance, the biosensor was applied to the determination of GSH in human serum samples through a standard addition method and determination of normal GSH concentration in original human serum samples with satisfactory results.