An experiment system for determining in vivo fluorescence of phytoplankton with five LEDs with different maximum emission wavelengths as excitation light sources was established. Based on the system,in vivo fluorescence of 10 different dilutions from the same water sample and 24 kinds of water samples from different sources was determined and the linear relationship of fluorescence intensity and chlorophylla concentration was also investigated. The results indicate that blue LED(maximum emission wavelength 442.7 nm) is the most proper excitation light source for determining in vivo fluorescence of phytoplankton,and limit of determination is 0.0278 μg/1(chlorophyll-a). Background fluorescence of DOM, especially the difference of phytoplankton species and fluorescence quantum efficiency, shows significant influence on the determination of in vivo fluorescence. Blue LED has a potential as excitation light source of phytoplankton fluorometry. In order to overcome the influence of phytoplankton species, fluorescence quantum efficient and DOM background fluorescence on in vivo fluorescence, further research should be carried out and proper adjustment methods are needed.