Phytoplankton concentration of functional reaction center is closely related to its growth environment and physiological state. A new method is used to analyze the phytoplankton concentration of functional reaction center based on the biological energy flow theory, in other words, based on the fluorescence kinetic parameters such as initial fluorescence efficiency (F0) and function absorption cross section (σPSII) is presented in this paper. The fluorescence parameters of Cholorella pyrenoidosa are measured under different conditions. The results show that there is a good agreement between the result of fluorescence kinetic parameter method and that of assimilation coefficient method under normal physiological conditions, and the correlation coefficient is 0.999. Compared with the assimilation coefficient method, the fluorescence kinetic parameter method is more accurate to analyze the changes in phytoplankton concentration of functional reaction center induced by photosynthetic activity (Fv/Fm) and photosynthetic unit sizes (nPSII) under abnormal physiological conditions. The phytoplankton concentration of functional reaction center obtained by the fluorescence kinetic parameter method is correlated with Fv/Fm, and the correlation coefficient is 0.920 under the short-term stress condition. The changes of nPSII caused by light can be measured by the fluorescence kinetic parameter method under the long-term light stress condition, which are consistent with the existing research results. This new method can accurately measure phytoplankton concentration of functional reaction center.