Using three-dimensional fluorescence spectroscopy technology, the relationship between three-dimensional fluorescence and toxicity of 5 strains of 4 microalgae species producing paralytic shellfish poisoning(PSP), which were Alexandrium minutum (Taiwan strain, AMSY), Alexandrium tamarense (Daya Bay strain, ATDY), Gymnodinium catenatum (Fangcheng Lane strain, GCFC), Alexandrium tamarense (Hong Kong strain, ATHK), Alexandrium catenella (South China Sea strain, ACSY), and 21 non-producing PSP microalgae species under different temperature conditions culture was studied. The results show that the toxin content of toxic algae per cell would change significantly under different temperature culture conditions and low temperature could promote the PSP production of toxic microalgae. By Db7 wavelet decomposition, the combined fluorescence spectrum of Ca3 scale components was selected as the characteristic spectrum and was used to establish the Fisher discrimination models, The combined fluorescence spectrum differences between toxic and non- toxic microalgae focused mainly on λ_ex 400~425, 450~545 nm and λ_em 715~750 nm bands. Using the Fisher discrimination models to dentification the toxic and non-toxic microalgae, the identification rate reached 93.7% and 93.3%, respectively, and the comprehensive discrimination rate was up to 93.5%. The method could be used to rapidly identify toxic microalgae in sea water and provide the theoretical basis for further developing toxic microalgae identification instruments.