In order to study the ultra-high pressure extraction of cordycepin from Cordyceps militaris fresh juice, the effect of opration conditions such as pressure, time, solid-liquid ratio and cycle number were analyzed. On the basis of single-factor test, the optimum extraction technology of cordycepin and mathematical regression model were established by response surface methodology. The result showed that the optimal extraction conditions were as followed: extraction pressure of 418 MPa, extraction time of 33 min, solid-liquid ratio of 12 (mV), cycle number of 1. Under these conditions, the measured value of dissolved quantity of cordycepin was 84.69 mg/g of extract, to which the relative standard deviation of predicted value (84.03 mg/g of extract) was 0.61%. The standardized regression equation can be used to predict the extraction yield of cordycepin from C. militaris fresh juice.