Spectrofluorometry of chlorella pyrenoidosa was studied by three dimensional excitation-emission (3DEEM) fluorescence spectroscopy and synchronous scan fluorescence spectroscopy with Δλ=20 nm in the stress of Hg2+, Cd2+, Cu2+ and Zn2+. The conclusion from two kinds of Spectrofluorometry was the same: after 96h stress by heavy metals, the maximum fluorescence values reduced obviously, chlorophyll-a and chlorophyll-b in the photosynthetic system were seriously damaged by heavy metal. Further analysis of the correlations between heavy metal concentration and fluorescence quenching efficiency I0/I can conclude that the toxicity of heavy metal and the fluorescence quenching efficiency I0/I were positively correlated, and they all increased with the heavy metal concentration and stress time. For one kind of heavy metals, synchronous scan fluorometry is a sensitive method for its toxicity assessment. Compared to 3DEEM fluorescence spectroscopy, synchronous scan fluorescence spectroscopy is less time consuming and of higher selectivity. It is suitable to assess the toxicity of pollutions in water.