The invention relates to a compound with cholesterol lowering activity extracted from burdock leaves and a preparation method and application thereof, can effectively solve the problem of extracting a new compound with lipid lowering activity from the burdock leaves and realize the application of the new compound in preparing lipid lowering medicaments. The preparation method comprises the steps of adding water into burdock leaves, conducting boiling and extraction, conducting concentration, feeding an obtained concentrate into an AB-8 column, conducting elution with water, 30% ethanol and 70% ethanol in sequence, collecting 70% ethanol eluent, recovering the solvent under reduced pressure, dispersing an obtained extractum by adding water, conducting extracting with n-butyl alcohol, recovering the solvent under reduced pressure, carrying out silica gel column chromatography, carrying out dichloromethane/methanol gradient elution, recovering the solvent under reduced pressure, carrying out secondary silica gel column chromatography, carrying out gradient elution with petroleum ether-ethyl acetate, collecting 100:70 eluate, recovering the solvent under reduced pressure, carrying out MCI column chromatography, carrying out methanol aqueous solution gradient elution, collecting 40% methanol eluate, recovering the solvent under reduced pressure, and carrying out reverse semi-preparation by using a YMC-Pack ODS-A column to obtain the compound with the activity of reducing cholesterol. The preparation method is easy to operate, high in guidance quality, high in separation speed and high in product purity.