Published studies showed that some contaminants were of time-dependent toxicity. Taking Chlorella pyrenoidesa as the test organism, 96-well microplates as experimental carriers, methanol (Meth), ethanol (Eth), dimethylsulfoxide (DMSO) and their mixtures with different volume ratios as research objects, the toxic effects of those organic solvents and their mixtures on C. pyrenoidosa in the exposure time of 0, 12, 24, 48, 72 and 96 h were investigated by the time-dependent microplate toxicity analysis. The results showed that the organic solvents obviously showed the time-dependent toxicity. The toxicity, toxicity order and toxicity increase rate of different organic solvents varied with the extension of the exposure time. Organic solvent mixtures also showed time-dependent toxicity with subtle differences, but their changing trends differed with the extension time. The toxicity of the mixtures with volume ratio of 1: 1, 1: 2, 1: 3 increased slowly within 0 - 24 h, while the toxicity of the mixtures with volume ratios of 2: 1, 3: 1 augmented quickly. The toxicity of the mixtures with ratios of 1: 1, 1: 2, 1: 3 fortified quickly within 24 -48 h, while that of mixtures with ratios of 2: 1, 3: 1 increased slowly. The toxicity increase rates of the five mixtures declined within 48 - 96 h. The toxicity changing trends of the organic solvent mixtures were influenced by the component with a high volume ratio.