Abstract: The Traditional Chinese medicine (TCM) has the characteristics of abundant resources, mild effect, and good anti-cancer effect, so research and development of anti-cancer drugs from TCM has become a hot spot. However, because the TCM extract has complex components and the pharmacological effects of TCM are usually a result of the cooperation of the multiple bioactive components binding to different biological targets, it was difficult to clarify the mechanism and foundation of the bioactivities of TCM. In this study, the Hela cell proliferation inhibition model coupled with centrifugal ultrafiltration mass spectrometry was established to screen the anti-cancer agents from TCM. Firstly, the antiproliferative effect of TCM extracts on the Hela cell lines was investigated by the MTT colorimetric method. And then, the promoter DNA of survivin gene and the protein calmodulin were selected as the cancer-related biological targets and the centrifugal ultrafiltration mass spectrometry method was used to screen and identify active components acting on the selected targets from strongest active extracts. The results showed that Catharanthus Roseus extract had the strongest inhibitory activity on Hela cell proliferation and the inhibitory activity was positively correlated to the concentration. Rhizoma Corydalis extract also had significant inhibitory activity on Hela cell proliferation at three different concentrations. Folium et Ramulus Cephalotaxi, Rhizoma Picrorhizae and Fructus Camptothecae Acuminatae extracts could significantly inhibit the Hela cell proliferation at high concentrations, while Acanthopanax Senticosus extract had no significant inhibitory effect on the Hela cell proliferation. Two binders of promoter DNA of survivin gene were screened from Catharanthus Roseus extract by centrifugal ultrafiltration mass spectrometry, and they were identified as serpentine and alstonine by multiple tandem mass spectrometry. Six binders of protein calmodulin were screened from Catharanthus Roseus extract and identified as serpentine, alstonine, tabersonine, leurosine, vincristine and 21’-oxo-leurosine. This study demonstrated the promising application of centrifugal ultrafiltration mass spectrometry in the screening of active components from TCM. The results could not only provide scientific references to the mechanism of the anti-cancer effects of the chemical components or the TCM extracts but also provide a rapid, sensitive and powerful platform for screening the bioactive components from TCM by using centrifugal ultrafiltration mass spectrometry.