Abstract: Water-soluble ZnSe quantum dots (QDs) were synthesized with L-glutathione (GSH) as stabilizer.These QDs were applied for ultrasensitive vitamin B₂（VB₂） detection.It mainly discussed the FRET system constituted by the water-soluble ZnSe (donor) and VB₂(receptor).The optimum conditions for FRET and established a VB₂ determination method based on the FRET principle were studied.The extent of fluorescence quenching (F₀/F) of ZnSe QDs was linearly proportional to the trace concentration of VB₂ from 1.54×10^-8 to 2.77×10^-7mol/L with a correlation coefficient of 0.9967,a detection limit (S/N=3) of 1.0×10^-8mol/L was obtained with the relative standard deviation of 1.2% (1.54×10^-7 mol/L,n=9).A novel method for selective detection of VB₂ by utilizing the prepared QDs as fluorescence probes has been developed,based on the quenching fluorescence of ZnSe QDs.The proposed method was successfully applied to the determination of VB₂tablets from three different factors.The results were satisfactory and the recoveries could fall in 99.28%-101.9% (n=6).The results were reliable,good agreement compare to those of UV-Vis absorption spectrometry.It was concluded that a novel and simple method based on fluorescence resonance energy transfer quenching theory was proposed for the determination of trace vitamin B₂.