Enzyme-assisted extraction (EAE) coupled with macroporous resin column chromatography (MRCC) was successfully used for the extraction and purification of cordycepin and adenosine from cultured Cordyceps militaris. After optimized by the response surface methodology (RSM), the extraction yields of cordycepin and adenosine were 86.45% and 70.06%, respectively. Subsequently, under the optimal separation conditions on NKA-II resins (loading 1 BV of extraction solution with pH 8.0, and eluting with 4 BVs of 70% ethanol at a flow rate of 3 BV/h), cordycepin (purity of 32.5%), and adenosine (purity of 39.9%) were obtained with an overall recovery rate more than 90%.