Forty-eight samples belonging to four common species of Cyanophyta are studied in order to reduce the chlorophyll a concentration measurement errors caused by unstable fluorescence spectra in vivo. We analyze the effects of algae species, growing period, and growing environment on the photosynthetic pigment composition and fluorescence efficiency by measuring the content of chlorophyll a and phycocyanin, as well as the three-dimensional fluorescence spectra of Cyanophyta in vivo. And we obtain the weight spectra by analyzing the spectral instability under different habitat conditions. The weight fluorescence spectrum of Cyanophyta in vivo is established based on the weighted average method. The measurement results that respectively obtained by the weight spectrum and different normalized spectra are compared, and the comparisons indicate that the weight spectrum can significantly reduce the dependence of fluorescence method on algae species, growing period, and growing environment, and thus increase the accuracy of chlorophyll a concentration of Cyanophyta. The relative error of the weight spectrum is 0.1%-30.4%, with a mean relative error of 12.8%. The maximum relative error of anabaena can be reduced by 104.1% in the weight spectrum.