An ulvan was extracted from green seaweed Ulva lactuca by ultrasound-assisted extraction. The extraction conditions were optimized by response surface methodology. The optimal conditions were extraction temperature at 84.75 °C, extraction time of 30.51 min, solvent to material ratio of 60.51 mL/g to achieve the yield of 22.5%. The ulvan is composed of repeated sequences of three disaccharides: →4)-β-D-Glucuronic acid(1→4)α-L-Rhamnose-3-sulfate(1→, →4)α-L-Iduronic acid(1→4)α-L-Rhamnose-3-sulfate(1→, and →4)α-D-Xylose-2-sulfate(1→4)α-L-Rhamnose-3-sulfate(→. The ulvan showed cytotoxic activities against five human cancer cell lines, including human hepatocellular carcinoma, human breast cancer, human cervical cancer, human colorectal adenocarcinoma and human undifferentiated thyroid carcinomas.