Flash extraction, ultrasonic extraction, and ultra-high pressure extraction technology were used to extract cordycepin from Cordyceps militaris residue medium. The extracting solution was filtered with ceramic membrane and cordycepin was separated and prepared by using high-speed countercurrent chromatography (HSCCC). The product structure was identified by LC-MS/MS. Antitumor activity of cordycepin was tested in mice S180 tumor. The results showed that ultra-high pressure extraction technology was better for cordycepin extraction. Combining a two-phase solvent system composed of ethyl acetate-n-butanol-water (2∶3∶5,V/V/V) with high-speed counter-current chromatography (HSCCC), the cordycepin was purified at 28℃ with flow rate of 3.0mL/min and revolution speed was 900r/min, while the upper phase was stationary phase and the lower phase was mobile phase. Cordycepin identified was identified by LC-MS/MS and the purity of cordycepin was 97.6%. About 181.90mg of cordycepin was obtained from 100g of Cordyceps militaris residue medium. The inhibition rate of cordycepin was 64.48% at doses of 100mg in vitro test.