Very close structure and property similarities among biothiols, such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), present a major challenge to achieve their discriminative detection. In this contribution, a nanomaterial surface energy transfer (NSET) system was established to discriminate GSH from Cys and Hcy with the photoluminescence (PL) “switch-on” response. The nanosensor was constructed using nitrogen and sulphur co-doped carbon dots (N,S-CDs) and silver nanoparticles (AgNPs) through assembling an energy transfer-based quenching system, featuring the pH-promoted distinct PL “switch-on” response. Under neutral conditions, only Cys and Hcy can encapsulate AgNPs, leaving little chance for N,S-CD binding on the surface of AgNPs, which can lead to PL signal recovery, and the total quantity of Cys and Hcy can be determined. However, at pH 3.0 all three kinds of biothiols can lead to the PL signal recovery and the total quantity of GSH, Cys, and Hcy can be determined, due to the similar slopes and intercepts of their calibration curves. Thus, the concentration of GSH could be further calculated and the strategy was successfully applied for the detection of GSH in human serum, demonstrating its potential in bioanalysis and significance in addressing biological and medicinal requirements.