Nitrogen-doped graphene quantum dots had been successfully synthesized and characterized by using transmission electron microscope, X-ray photoelectron spectroscopy, absorbance spectrum, fluorescence emission spectrum, and fluorescence decay curve. TEM results indicated that the diameters of the as-prepared nitrogen-doped graphene quantum dots were in the range of 2 - 5 nm and the lattice space is about 0.276 nm; Raman spectrum result indicated that there were two characteristic peaks, generally named D (~1408 cm−1) and G (~1640 cm−1) bands; both TEM and Raman spectrum results indicated that the as-synthesized product was graphene quantum dots. Deconvoluted high resolution XPS spectra for C1s, O1s, and N1s results indicated that there are -NH-, -COOH, and -OH groups on the surface of nitrogen-doped graphene quantum dot. Fluorescence emission spectrum indicated that the maximum fluorescence emission spectrum of nitrogen-doped graphene quantum dots was blue shift about 30.1 nm and the average fluorescence decay time of nitrogen-doped graphene quantum dots increased about 2 ns, compared with graphene quantum dots without doping of nitrogen. Then, the as-prepared nitrogen-doped graphene quantum dots were used to quantitatively analyze brilliant blue based on the fluorescent quenching of graphene quantum dots, and the effect of pH and reaction time on this fluorescent quenching system was also obtained. Under selected condition, the linear regression equations were F0/F=0.0087 (brilliant blue) + 0.9553 and F0/F=0.01205 (brilliant blue) + 0.6695, and low detection limit was 3.776 μmol/L (3.776 nmol/mL). Once more diluted N-GQDs (0.05 mg/mL) were used, the low detection limit could reach 94.87 nmol/L. Then, temperature-dependent experiment, absorbance spectra, and dynamic fluorescence quenching rate constant were used to study the quenching mechanism; all results indicated that this quenching process was a static quenching process based on the formation of complex between nitrogen-doped graphene quantum dots and brilliant blue through hydrogen bond. Particularly, this method was used to quantitatively analyze the wine sample, of which results have a high consistence with the results of the spectrophotometric method; demonstrating this fluorescence quenching method could be used in practical sample application.