Prorocentrum dentatum, Gymnodinium stein and Skeletonema costatum are the main algae which frequently cause red tide. In this paper in vivo fluorescence excitation spectra were measured and the discrimination method of the red tide algae was established by the similarity index of spectra. The results show that the in vivo fluorescence excitation spectra were well reproduced (RSD≤5%) and the spectra precision was very high (RSD≤3%). The similarity index between the standard spectra of one algae species and hypothetical mixed spectra of algae and spectra of mixed algae assemblage was calculated. There is a linear relationship between the similarity index and the relative content of the algae. So the red tide algae can be discriminated by calculating the similarity index between fluorescence spectra of mixed algae and their standard fluorescence spectra, and the relative content of the red tide algae can also be detected semi-quantitatively. There is almost no influence of noise on the discrimination of red tide algae when noise is lower than 25%.