To separate and select anti-tumor activity part from the leaves of Paliurus ramosissimus. Methods: Extracts from the leaves of Paliurus ramosissimus were extracted with petroleum ether, ethyl acetate and n-butyl alcohol respectively. In vitro anti-tumor experiments were performed at the three extract sites. The optimal site with best anti-tumor activity then was screened by silica gel column chromatography, and MTT assay was used for the comparison of components obtained for antitumor activity. Results: In vitro anti HepG-2 cells and HeLa cell experiments showed that ethyl acetate fraction extracts had good anti-tumor activity. Separated by a silica gel column, five components were collected by MTT assay, which found that when the ratio of petroleum ether: ethyl acetate were 1∶1 and 1∶2, both eluted fraction group had in vitro anti-tumor activity and when the ration of the elution of petroleum ether to ethyl acetate was 1∶2 the anti-tumor function was better. IC50 of HepG-2 cells and HeLa cells were 45.79 and 13.16 μg·mL-1 respectively. Conclusion: Two kinds of active ingredients with anti-tumor effect were separated from the leaves of Paliurus ramosissimus by ethyl alcohol, which may provide guidance for the further separation of anti-tumor active ingredient.