Microalgal fluorescence kinetics method, as a promising technique for rapidly detecting water toxicity, still lacks a consistent response index with sensitive response characteristics to different types of toxic pollutants. In order to solve this problem, in this study, the influences of six typical toxic pollutants belonging to different types, including the heavy metal cadmium, the herbicide atrazine, organic chemical raw materials 1,4-benzoquinone and 1,4-dichlorobenzene, and the disinfection by-products chloroacetic acid and trichloroacetonitrile on the fluorescence rise kinetics (OJIP) curve of microalga Chlorella pyrenoidosa were investigated. According to the same characteristics of changes in the OJIP curves of different toxic pollutant treatments, a new response index for detecting the comprehensive toxicity of water (RI_ct) was constructed. By comparing with the maximum photochemical quantum yield of photosystem II (PSII) (F_v/F_m), the potential quantum yield of PSII (F_v/F_o), the performance index of PSII (PI_ABS) and the quantum yield of electron transport of PSII (φ_Eo), the feasibility and sensitivity of quantitative response of RI_ct to the toxicity of each pollutant and polluted water containing multiple pollutants were verified. The results indicated that all the six toxic pollutants had the same influence on parameters F_v(m-j)/F_o and F_v(m–o)/F_o contained in the OJIP curve, and RI_ct constructed based on F_v(m-j)/F_o and F_v(m–o)/F_o had a good logistic curve concentration–response relationship with each pollutant. By comparison, the response sensitivity of RI_ct to all pollutants was significantly superior to F_v/F_m, F_v/F_o and φ_Eo, and also had better stability in the toxicity response to each pollutant compared to PI_ABS, indicating that RI_ct was a suitable response index for rapidly and sensitively detecting water comprehensive toxicity based on microalgal fluorescence kinetics. This study provides an important basis for the development of rapid and sensitive detection methods for water toxicity.