Rutin, an important kind of flavonoid, has some physiological functions, including antitumor activity, anti-diabetic, anti-oxidation, etc. Thus, it is important to develop a novel method for the simple and sensitive determination of rutin. A simple, highly sensitive and highly selective fluorescence sensor has been established to detect rutin based on glutathione-stabilized copper nanoclusters (GSH-CuNCs). GSH-CuNCs with excellent luminescent properties were prepared using glutathione (GSH) as a stabilizer and ascorbic acid (AA) as a reductant. Ultraviolet absorption spectra, fluorescence excitation and emission spectra studied the optical properties of GSH-CuNCs. The results showed that the GSH-CuNCs have strong fluorescence emission at 420 nm with an excitation wavelength of 365 nm. The absorption spectra showed that the GSH-CuNCs have an obvious absorption peak at 293 nm (UV region), but no absorption was observed above 400 nm (visible region). It was indicated that the GSH-CuNCs had molecular-like properties and did not have larger copper nanoparticles, suggesting the high purity of GSH-CuNCs. Moreover, the fluorescence intensity (at 420 nm) of GSH-CuNCs remained at about 96% after being stored at 4 ℃ for 3 months. When rutin was present, the fluorescence intensity of GSH-CuNCs was significantly quenched. It was ascribed to the absorption spectra of Rutin overlapped largely with the fluorescence excitation spectra of GSH-CuNCs, which led to the inter-filter effect (IFE). The effect of pH and reaction time for the detection of Rutin was optimized. It was found that the quenching effect was the best when pH was 7.5 and the reaction time was 10 minutes. Under optimum experimental conditions, the fluorescence emission spectrum of GSH-CuNCs was recorded with different concentrations of Rutin. The results confirmed that the sensor had a good fluorescence response to Rutin, with a linear range of 1.00～200 nmol·L-1 and a limit of detection of 0.300 nmol·L-1. Different interfering substances with the same concentration were introduced into the sensing system. It was observed that the fluorescence intensity of GSH-CuNCs could be quenched only in the presence of Rutin, which implied that this method has a good selectivity for determining Rutin. The method has been applied to detect rutin content in buckwheat tea samples. This assay was no modification, simple and convenient, and with less sample consumption has good sensitivity.