Chlorophyll fluorescence emission characteristics of phytoplankton in vivo is closely related to the exciting wavelength and intensity. The fluorescence induct curves under 4 wavelengths of LED (450, 525, 590, 620 nm) and various intensities using chlorella vulgaris and microcystis aeruginosa are tested. Research results show that smooth induct curve are obtained when corresponded exciting light intensity are below 2.8, 4.6, 3.8 4.3 μmol/(m-2·s). Fluorescence intensities of Fo are recorded at the same gain setting, which represent the pigment level. A discrete spectrum can be obtained when considering exciting light photosynthesis active radiation (PAR). Fluorescence affected by wavelength is analyzed combining absorbance data. Significant different chlorophyll fluorescence induct curves are got between phytoplankton in vivo, extracted by 90% ethanol and addition with 0.4 μmol/L 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU). This work can provide theoretical and data support for phytoplankton photosynthesis activity measurement.