The fluorescence spectra of synthetic food dyes of sunset yellow and tartrazine are analyzed. The fluorescence peak wavelengths of sunset yellow and tartrazine are 576 and 569 nm, respectively, while the fluorescence spectra widths are 480–750 and 500–750 nm induced by ultraviolet light between 310–400 nm. The fluorescence spectra of sunset yellow overlap heavily with those of tartrazine, so it is difficult to distinguish them. Based on the principle of radial basis function neural network, a neural network is obtained from the training of the 14 groups of experimental data. The results show that the species of sunset yellow and tartrazine could be recognized accurately. This method has potential applications in other synthetic food dyes detection and food safety inspection.