Herein, 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 Cu ²⁺ toxicity. The results are as follows: the inhibition effect of F₀, Fm, Fv, Fv/Fm, Yield, rP, JVPⅡ, α and Ek on Cu ²⁺ toxicity is significant, and yield, rP, α and Fv/Fm could be used as the evaluation indexes for Cu ²⁺ toxicity analysis at 24 h; the inhibitory effect of Yield, rP, α and Fv/Fm has a good dose-effect relationship with Cu ²⁺ toxicity. The fitting correlation coefficient R² of the logistic function is 0.9989, 0.9992, 0.9991 and 0.9977, and the resulting EC_50-24h is 61.05, 66.31, 69.41 and 99.61 μmol·L ^-1, respectively. The parameters Yield, rP and α have higher fitting correlation coefficients than the conventional parameter Fv/Fm, and their EC_50-24h values decrease by 38.7%, 33.45% and 30.3%, respectively. The experimental results could provide reference for the detection of biological toxicity based on photosynthetic inhibition effect of algae.