A glutathione (GSH) optical sensor with high sensitivity and exceptional selectivity was established for the first time. Zeolitic imidazolate framework-8 (ZIF-8) as a model was used for the first time to entrap Mn²⁺:ZnS quantum dots (QDs) and a rhodamine B derivative (RBD) by self-assembly. Benefiting from the confinement effect of ZIF-8, the loaded QDs and RBD can be brought into close proximity for energy transfer to occur. In the presence of GSH, the fluorescence resonance energy transfer (FRET) process from the QDs to RBD can be initiated, rendering the fluorescent nanoprobe to exhibit a response to GSH. The fluorescence intensity of Mn²⁺:ZnS@ZIF-8@RBD decreased with an increase in the GSH concentration in the linear range of 5–120 μM and a detection limit of 1.5 μM. This finding leads to a method for the fluorescence detection of GSH with excellent selectivity over other reactive thiols. Moreover, because of its good accuracy and excellent recovery, the nanoplatform can be applied for GSH sensing in real human serum and urine samples. Hence, the developing probe may be extended to other optical sensing domains or drug carriers and has tremendous potential in the field of biomedicine.