Developing an effective detection method for benfluralin (BFA) is of great significance, since BFA as most widely used herbicides can be bioaccumulated by aquatic organisms in environment, possessing potential risks to human health. Owing to aggregation-caused quenching effect, most fluorescent detection methods based on donor–acceptor organic fluorophores suffered from very low sensitivity towards BFA in water system, hampering the bioimaging application in plants. In this work, we reported a novel surfactant-assisted fluorescent probe enabling detection of BFA in water with a high sensitivity. The involvement of specific surfactant Triton X100 (TX100) could amplify the response signal of probe more than 100-fold. The detection limit for BFA was determined to be 80 nM, satisfying the environmental protection requirements. Moreover, we demonstrated applications of this strategy for the fluorescent imaging of BFA in plant. The absorbance of BFA into roots of Arabidopsis thaliana and castor seedlings was successfully observed based on this method.