The best dark adaptation time of Chlorella pyrenoidosa, Snedesmus obliquus and Microcystis aeruginosa stressed by Cu2+ was investigated by chlorophyll fluorescence measurements. Photosynthetic fluorescence parameters were measured under light and dark conditions. Measurement time in light condition were 30 s, 1 min, 3 min, 5 min, 10 min and 20 min as well as in dark condition. Significant difference of dark adaptation time was analyzed mainly based on the photochemical quenching parameter and t-test method. The results show that compared to light, the potential maximum quantum efficiency values of three algaes increase slightly under dark adaptation and the yield value remaines the same; the photochemical quenching parameter value and non photochemical quenching parameter value increase significantly as time goes on; the maximum photochemical quenching parameter value of Microcystis aeruginosa achieves when the algae is treated under light in 1 min, and Microcystis aeruginosa needs no dark adaptation may because blue-green algae state conditions shifts under dark adaptation; the dark adaptation time of algaes are not better by long time treated, and the best dark adaptation time of Chlorella pyrenoidosa and Snedesmus obliquus are 5 min and 10 min respectively. All of these will provide a reliable basis for further studies on the inhibiting effect of toxicant on algaes.