In vivo chlorophyll fluorescence excitation spectra at 675 nm emission wavelength of six phytoplankton species belonging to dinophyta and bacillariophyta were studied. The fourth-derivative analysis was performed on the spectra to set up the uniform initial central wavelengths for excitation spectra of dinophyta and bacillariophyta, respectively, according to the position and number of maxima on the derivative spectra. With these determined wavelengths as centre, Gaussian decomposition and multi-peak fitting were done to the spectra to establish Gaussian database on the level of divisions. The results suggested that at the excitation wavelength from 350 to 550 nm, significant differences of Gaussian database presented between dinophyta and bacillariophyta while similar characteristics were found in the same division. The fitting curve reconstructed from Gaussian parameters was in coincident with the in vivo excitation spectra. Gaussian decomposition could reproduce overlapping pigment fluorescence peaks. This method provides not only a useful tool to differentiate dinophyta and bacillariophyta, but also a new technique for determination of in vivo photosynthetic pigments.