The mixed solutions of brilliant blue and indigotine are prepared and the fluorescence spectra of them are experimentally measured. The serious overlapping spectra of brilliant blue and indigotine are solved by means of the first-derivative fluorescence spectrometry. The wavelet coefficients, obtained by compressing the spectral data using wavelet transformation (WT), are taken as inputs to establish the radial basis function neural network (RBFNN). The neural network model can realize simultaneous determination of brilliant blue and indigotine, and the mean relative errors of both compounds are 1.84% and 1.26% , respectively. Synthetic food dyes are extensively used in food processing to improve the sensory quality of food products [1] . However, the raw material of synthetic food dyes is mostly coal tar, and synthetic food dyes may be degraded into carcinogens by azo reductases in human body [2] . Therefore, it is necessary to determine the synthetic food dyes accurately in food safety supervision.In our previous work, a single synthetic food dye, such as Ponceau 4R [3] and Amaranth [4] , can be determined using conventional fluorescence spectrometry. But in practice, synthetic food dyes are often mixedly used to get an ideal color. Due to the probable overlapping of fluorescence spectra of different dyes, the conventional fluorescence spectrometry is not accurate enough to simultaneously determine the multicomponent dyes.In this paper, brilliant blue and indigotine, which have serious overlapping fluorescence spectra, are chosen as experimental subjects. And the derivative fluorescence spectrometry is introduced to solve the overlapping spectra. Finally, combined with WT and RBFNN, a prediction model is established to determine these two dyes simultaneously.The samples are brilliant blue and indigotine standard solutions, each 1 mg/ml, provided by the National Research Center for Certified Reference Materials. We use ultrapure water to dilute the standard solutions and prepare 24 different concentrations of mixed solutions with a range of 0-20 g/ml for both brilliant blue and indigotine. The spectra of mixed solutions are measured on SP-2558 multi-function spectral measurement system, the excitation wavelength is 300 nm, and the range of emission wavelength is from 350 nm to 800 nm.We take 5 g/ml brilliant blue solution, 5 g/ml indigotine solution and the mixed solution with a concentration of 5 g/ ml brilliant blue and 5 g/ml indigotine for instance, and the fluorescence spectra of them are shown in Fig.1. It is obvious that the spectra of these solutions overlap severely, among which the fluorescence peaks of brilliant blue are located at 444 nm and 665 nm, while those of indigotine are located at 416 nm and 678 nm. And it is apparent that the spectrum of the mixed solution is not a simple superposition of the spectra of brilliant blue and indigotine, due to the competition between them. Meanwhile we can see that the brilliant blue has an edge in fluorescence competition, which causes the similarity between ...