To identify ichthyotoxic microalgae species and its hemolytic activity by three-dimensional fluorescence spectra, the relationship between hemolytic activity and the three-dimensional fluorescence of typical ichthyotoxic algae (Karenia mikimotoi, Chattonella marina, Chattonella ovata) cultured in different Fe2+ concentrations was investigated with methods of Coif2 wavelet and Fisher discriminant analysis. The results showed that the differences of three-dimensional fluorescence spectra between ichthyotoxic algae and non-ichthyotoxic algae were within the 1 to 10 data points (wavelength λem=650～680 nm) and 35 to 47 data points (wavelength λem=725～750 nm, λex=400～425 nm). Fisher discriminant analysis showed that the correct discrimination ratios of ichthyotoxic algae and non-ichthyotoxic species are 91.7% and 100%, respectively, and the ratio of algae with medium hemolytic activity (≥10 HU, <20 HU) was 70% and that of algae with low hemolytic activity (<10 HU) and high hemolytic activity (≥20 HU) are both of above 80%.