Chitooligosaccharides (COS) was obtained from chitosan by depolymerization with enzyme and analyzed by HPLC and TOF-MS, and the results indicated that the polymerization degree of COS was 2–18. In order to explore the anti-angiogenic activities of COS, the effect of COS on chicken chorioallantoic membrane (CAM) angiogenesis and on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) induced by human hepatoma carcinoma cells (HCC) culture fluid was measured. The results showed COS had no toxicity to normal HUVECs, but could inhibit the CAM angiogenesis and the proliferation, migration and tube formation of induced HUVECs. All of these results indicate that COS have potential anti-angiogenic activities and can counteract the stimulation of HCC-culture-fluid on endothelial cells at a certain level.