Fluorescence spectroscopy had the advantages of high sensitivity and fast detecting. The three-dimensional(3D)fluorescence spectrometry was more informative, better selectivity, more advantage than the general fluorescence spectroscopy on the multicomponent separation. The determination method of chlorophyll-a in water based on the discrete three-dimensional fluorescence spectrometry in vivo was studied. It extracted different fluorescence fingerprint properties of five divisions of algae (Cyanophyta, Chlorophyta, Bacillariophyta, Dinophyta and Cryptophyta), measured chlorophyll-a concentration of every algae and got the total chlorophyll-a concentration by plus. The three-dimensional fluorescence algae in-situ analyzer (AFA) was manufactured based on it. The chlorophyll-a concentration in Lake Taihu was measured by AFA. The measured values were compared with YSI, BBE and the spectrophotometry. It showed no significant difference between AFA and the spectrophotometry. There were good correlation among AFA, spectrophotometry and BBE. The correlation coefficient was above of 0.96. AFA was better precision and accuracy than the general fluorescence spectroscopy. It was a fast and effective in-situ method.