Chlorella pyrenoidosa, Microcystis aeruginosa and Scenedesmus obliqnus were selected as sensitive algae in this experiment. The effect of microalgae Phtosynthesis characteristic was studied after exposure of 4 kinds of herbicides (Atrazine, Diuron, Propanil and Bentazone) and its mixture. Meanwhile, the best response time of microalgae fluorescence to the herbicides was determined in this study. The results showed that 4 selected herbicides significantly reduced the algal cells in the thylakoid membrane of light system Ⅱ maximum photosynthetic efficiency (Fv/Fm), the actual photosynthetic efficiency (Y(Ⅱ)), the absolute electron transport rate (ETR) and photochemical quenching coefficient (qP), however, increased non-photochemical quenching coefficient (qN) with the increasing herbicide dosages. The Chlorella pyrenoidosa at 400 μg·L-1 Diuron had the largest decrement of Fv/Fm(41%). Algae all displayed the effects of different degree on photoinhibition in conditions of 4 kinds of herbicides individual and mixing stressing, with the decrease of photosynthetic efficiency, electron transfer rate and photosynthetic activity, but increase of light protection ability. Algae itself had a certain light protection mechanism that could reduce the influence of herbicides stress. Herbicides affected the intensity of chlorophyll fluorescence, in which the effect of Bentazone on Chlorella pyrenoidosa was the most significant. The intensity of Chlorella pyrenoidosa chlorophyll fluorescence at 400 μg·L-1 Bentazone had the largest decrement (44%).