The invention provides a preparation method of Rhizoma Anemones Raddeanae extract. The method comprises the following steps: drying and dehydrating Rhizoma Anemones Raddeanae, carrying out ultrafine crushing, adding ethyl acetate, extracting Rhizoma Anemones Raddeanae, filtering, collecting filter residues, extracting the filter residues with ethanol, filtering, taking the obtained supernatant, carrying out reduced pressure concentration, adding a methanol-water solution, carrying out ultrasonic dissolving, carrying out separation and purification in a C18 reversed-phase chromatographic column with an A ultrapure water and B methanol two-component mobile phase under the following separation conditions: 95% A isocratic elution in 0-5min, 87% A isocratic elution in 5-15min, 40% A isocratic elution in 15-25min, 30% A isocratic elution in 25-35min, 25% A isocratic elution in 35-45min and 100% B isocratic elution in 45-55min, collecting a 47-48min eluate, carrying out reduced pressure concentration, and drying the finally obtained concentrate to obtain the target product Rhizoma Anemones Raddeanae extract. The separation method has the advantages of stability, high repeatability, large preparation quantity, simple operation, time saving, and suitableness for industrial large-scale production.