A new and environmentally friendly approach for the preparation of glutathione-stabilized silver–gold nano-alloys (GSH-AgAuNAs) with high fluorescence was proposed in this work. The as-prepared GSH-AgAuNAs were characterized with various methods and coupled with copper ions (Cu²⁺) to form a fluorescent switch probe (GSH-AgAuNAs/Cu²⁺ combination) for the detection of L-histidine. The fluorescence of the GSH-AgAuNAs was first quenched by adding an appropriate amount of Cu²⁺ solution. Then, in the presence of L-histidine, the GSH-AgAuNAs/Cu²⁺ combination solution exhibited an obvious fluorescence enhancement due to the specific interaction between the imidazole group of L-histidine and Cu²⁺. The strong chelation between L-histidine and Cu²⁺ illustrated the feasibility of constructing a selective “switch on” probe for the detection of L-histidine over other amino acids. Different from other methods for detecting L-histidine based on fluorescent nanomaterials, our work promises high selectivity, simplicity and the avoidance of organic solvents. Under the optimal conditions, the newly constructed GSH-AgAuNAs/Cu²⁺ fluorescent probe showed a satisfactory linear detection range of 2 to 40 μM, with a detection limit of 1.19 μM. The practical use of the GSH-AgAuNAs/Cu²⁺ combination in real human serum samples was tested, illustrating its potential application in L-histidine detection.