Three-dimensional fluorescence spectrometry described the relationship between fluorescence intensity and the wavelength of emission and excitation. More information of fluorescence spectrum can be provided. Characteristic fluorescence spectrum can be obtained by dividing data matrix of three-dimensional fluorescence spectra from complex mixture by the parallel factor analysis (PARAFAC) model, which can separate the spectrum from different kinds of fluorescence matters. By means of PARAFAC model and three-dimensional fluorescence spectroscopy, the determination of four components with overlapping spectra is described. It has been tested satisfactorily for the analysis of two binary and a quadruplex mixtures consisting of phenanthrene, anthracene, pyrene and fluoranthrene.