Abstract: The purpose of this study was to determine the rapid extraction process of the active ingredient from burdock leaves and its inhibition performance of wood-rot fungus. On the basis of the preliminary experiment with the Coriolus versicolor as indicator fungi and the single factor experiments with the extraction rate as an indicator, the response surface method (RSM) was used to optimize the extraction of active constituents from the leaves of Arctium lappa, and the anti-fungal ability to wood rot fungi was determined. And the active constituents were separated and identified by U-HPLC-MS under the optimization condition of extraction. Experiment results showed that: the anti-fungi property with acidic water extraction was superior to other ways in the preliminary experiments. After four factors and three levels of response surface optimization, the extraction rate reached 45.81% under the following conditions: the ratio of liquid to material was 30 mL/g, the extraction temperature was 52 ℃, extraction time was 77 min, and the solvent pH was 2.0. The average extraction yield was 44.86% verified under the optimization condition, and the theoretical value difference of the model was -2.08%. The effect of the extract on inhibiting white rot fungi was 16.47% higher than that of the pre-experiment with the acidic water. Twelve kinds of anti-fungi active compounds were isolated and identified by U-HPLC-MS, i.e. organic acids (quinic acid, protocatechuic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, dimer chlorogenic acid, chlorogenic acid, 4, 5-dicaffeoylquinic acid), flavonoids (arctinone-a), glycosides (rutin, 5′-propanediolmatairesinoside), terpenes (β-eudesmol). The minimum inhibitory concentrations of the extracts obtained by this method were 1.88% and 2.31% for white rot fungi (Coriolus versicolor) and brown rot fungi (Gloceophyllum trabeum), respectively.