Abstract: In order to determine carmine concentration in the mixed pigment solution, normalization method was used to preprocess the fluorescence spectra. The processed data were combined with radial basis function neural network to establish the prediction model of carmine content. The average relative error of the prediction results of 3-D synchronous fluorescence spectrometry and 3-D ordinary fluorescence spectrometry were 2.86% and 11.12% respectively. The results showed that the 3-D synchronous fluorescence spectrometry was superior for the determination of the mixed pigment solution. The research provides the help for the prediction of the pigment concentration in the mixed pigment solution.