Abstract: Semi-preparative HPLC was used to isolate and purify 3'-O-methyl-ellagic acid-4-O-琢-L-rhamnopyra鄄 noside from the root of Mallotus apelta. Its chemical structure was elucidated by 13C-NMR and 1H-NMR; 10 batches of the roots of Mallotus apelta samples from different sources were analyzed by HPLC gradient elution method. The results showed that the purity of 3'-O-methyl-ellagic acid-4-O-琢-L-rhamnopyranoside reached 98.50% by using area normalization method, 15 common peaks were selected in chromatograms, all the common peaks were separated effectively, and 10 batches of the roots of Mallotus apelta fingerprint had good similarity. The results indicated that semi-preparative HPLC was a simple, quick and efficient method to prepare 3'-O-methyl-ellagic acid-4-O-琢-L-rhamnopyranoside. Fingerprint analytical method, being with good precision and repeatability, could be useful in the quality control of the root of Mallotus apelta.