Abstract: Tetranychus urticae is one of the major threat in global agricultural production, whereas the currently commercially available acaricides are limited in variety. Trifluenfuronate, as a novel acaricidal candidate, has a mode of action that remains unclear to date. Herein, we employed theoretical target-fishing methods, including molecular similarity analysis, pharmacophore mapping, and reverse molecular docking, to systematically predict the potential targets of trifluenfuronate in T. urticae. Firstly, molecular similarity searches were performed against the SwissTargetPrediction and PADFrag databases. Subsequently, reverse screening through pharmacophore model matching was carried out using PharmaDB and PharmMapper. Finally, reverse molecular docking against the full proteome of T. urticae was carried out via Discovery Studio to prioritize high-affinity target proteins. The results suggest that the acaricidal mechanism of the novel acaricide trifluenfuronate against T. urticae may be related to its interference with targets associated with energy metabolism and signal transduction. In summary, our findings have laid a robust theoretical foundation for elucidating the mode of action of trifluenfuronate and accelerating the rational design of novel acaricidal agents.