In order to improve the sensitivity of the algal chlorophyll fluorescence method for the detection of trace herbicides, the optimal response time of microalgae to the toxicity of three triazine herbicides (Atrazine, Prometryn and Terbumeton) was investigated, as well as the sensitization effect of methyl-ethyl vegetableoil on the biological toxicity of herbicidesdetected by algal fluorescence method were further studied. In addition, four chlorophyll fluorescence kinetic parameters of Fv/Fm, Y(Ⅱ), NPQ and qP were measured by WATER-PAM-WALZ as the toxicity evaluation index, and Chlorella proteinacea, Microcystis aeruginosa and Scenedesmus obliquus were adopted as the test algae. Therefore, the results were as follows: ①The optimal biotoxic response times of Chlorella proteinosa, Microcystis aeruginosa, and Scenedesmus obliquus were to the three herbicides 20, 10 and 10 min, respectively. Moreover, the most effective concentrations of methyl-ethyl vegetable oil were 0.25%, 0.5% and 0.25%, respectively. ②Scenedesmus obliquus had the highest sensitivity to the three herbicides, while Microcystis aeruginosa was more adaptable and resistant to herbicides. Under 20 μg·L-1 Prometryn exposure, the Fv/Fm inhibition rates of both Scenedesmus obliquus and Chlorella proteinosa were up to 23%, while that of Microcystis aeruginosa only reached 8%. ③Y(Ⅱ) and NPQ showed the highest sensitivity to herbicide toxicity under the treatment of methyl-ethyl vegetable oil. Besides, the inhibitory effect of 40 μg·L-1 Prometryn on Y(Ⅱ) of Scenedesmus obliquus was 56%, and the inhibition rate of Fv/Fm was up to 41%, which was 2.5 times that of the control group. However, when exposed to 200 μg·L-1 herbicide, the NPQ values of the three algae increased by more than 1.5 times, and the qP was less affected by methyl-ethyl vegetable oil. Hence, the above results demonstrated that the contact area between herbicide and algae cells was enlargedby methyl-ethyl vegetable oil, and the penetration of herbicide was enhanced, which improved the toxic response of algae fluorescence to herbicide as well as reduced the detection limit of herbicide. In this study, we first utilized methyl-ethylated vegetable oil to detect triazine herbicides using the algal fluorescence method. The results demonstrated that the algal fluorescence method exhibited a highly effective detection capability when combined with methyl-ethylated vegetable oil. This approach successfully addressed the issue of insensitive responses observed in chlorophyll parameters in certain cases. As a result, a novel and rapid method for sensitively detecting trace triazine herbicides biotoxicity in the environment has been established.