The development of cyanobacterial biomass detection technology is an important part to deal with the frequent occurrence of water bloom events. Phycocyanin, to some extent, as a special protein of cyanobacteria, is more accurate reaction in natural water cyanobacterial biomassthan chlorophyll, thus become an important index in cyanobacterial biomass detection. This paper, with different light intensity and different growth stages as the research objects. compares the reliability of the two methods of the envelope method and the single point method , exploring the fluorescence spectral characteristics of phycocyanin in vivo Microcystis Anabaena by using three-dimensional fluorescence spectroscopy. The results show that: (1) The fluorescence intensity increases with the growth of long-term; (2) It is more reliable to use envelope method to analyze the characteristic fluorescence spectra of the fluorescent spectrum than the single point method. (3) The fluorescence spectra of Phycocyanin in vivo Microcystis were basically unchanged in EX=614 nm/ EM=654 nm. The excitation wavelength of the phycocyanin fluorescence in vivo Microcystis decreases with the growth with 610 and 620 nm in the long term, and the emission wavelength varied between 650 and 660 nm, which is related to the sample particle size and spectral scanning mode. This study provided the experimental basis for the development of the fluorescent detection technology of cyanobacteria.