An efficient method for simultaneous detoxification and preparative separation of chlorogenic acid from Eupatorium adenophorum was developed. The entire procedure consisted of two separation steps. The first step was enrichment of chlorogenic acid from the crude extract by NKA-II macroporous resin. The purity of chlorogenic acid increased from 5.3% to 20.9% with a recovery of 88.7%. Subsequently, polyamide resin was used to achieve a higher purity. After the polyamide resin treatment, the main toxin 9-oxo-10,11-dehydroageraphorone was removed and the purity of chlorogenic acid reached 86.5% with a recovery of 84.8%. It provided a new way for the utilization of E. adenophorum.