As single-celled organisms, algae were small in size, easy to culture, sensitive to poisons, and able to observe toxic symptoms at the cellular level. They were ideal test organisms for rapid detection of biotoxicity in water quality. However, “algal growth inhibition test” relied on the reproductive and metabolic process of algal cells, and the measurement cycle was long, so it could not meet the needs of rapid detection of biological toxicity. The response speed and sensitivity of algae’s photosynthetic process to the toxicity of pollutants were significantly better than that of the “algae growth inhibition test”. The variable fluorescence Fv or maximum photochemical quantum yield Fv/Fm were mostly used as the endpoint of the biotoxicity reaction in the existing “photosynthesis inhibition experiments”, and the lack of comparative analysis on the sensitivity of response of multiple photosynthetic fluorescence parameters led to the low sensitivity of quantitative biotoxicity detection. Planktonic algae were ideal biotoxic test subjects, and Chlorella pyrenoidosa was taken as the subject, and photosynthetic fluorescence parameters of algae were used as toxicity evaluation indexes to study the response law of multiple photosynthetic fluorescence parameters under DCMU toxicity, to improve the speed of DCMU biological toxicity test and sensitivity, in this paper. The results showed: (1) The photosynthetic fluorescence parameters F0, Fm, σPSⅡ, τQA, Fv, Fv/Fm, Yield, rP, NPQ, α under DCMU toxicity were significantly responsive, and the responses of α, rP, Fv/Fm, Yield, NPQ in 5 minute were similar to the responses in 96 hour. (2) The inhibitory effect of α, rP, Fv/Fm, Yield, NPQ had a good dose-effect relationship with DCMU concentration. The correlation coefficient of parameter NPQ and EC50 were 0.998 5 and 2.41 μg·L-1, respectively, which are significantly better than the other four parameters and the measurement result of 96 hour. If the photosynthetic fluorescence parameter NPQ was used as the endpoint of 5 min quantitative measurement of DCMU biotoxicity, then the detection time of DCMU biotoxicity detection could be greatly shortened (from several hours to 5 min) and the detection sensitivity could be significantly improved (EC50 was 81.4% lower than the result which was calculated through the conventional parameter Fv/Fm). The experimental results provided basic data for the quantitative detection of DCMU biotoxicity based on the photosynthetic inhibition effect of algae, and the research method also provided a reference for the screening of photosynthetic fluorescence parameters of algae under the stress of other pollutants.