Use of microwave radiation is one of the most potential techniques in polysaccharide derivatives synthesis due to its advantages such as higher yields, milder reaction conditions and shorter reaction times. This study was aimed at producing sulfated polysaccharides by microwave irradiation using polar reagents and solvent. Six sulfated Artemisia sphaerocephala polysaccharide derivatives (SPAS) were obtained by using chlorosulfuric acid/pyridine method in the reaction duration range of 15-300 min at a fixed microwave power of 100W. Synthesis of SPAS under microwave filed was highly effective and characterized by time saving and high degrees of substitution (DS). The chemical structure of SPAS was confirmed by FT-IR and 13C NMR that sulfation had occurred and the substitution position was mainly at C-6. Size-exclusion chromatograph combined with multi-angle laser photometer (SEC-MALLS) data indicated that short reaction time minimized acid-catalyzed degradation of polysaccharide chains. AFM observation demonstrated that low-MW derivative exhibited aggregation of polysaccharide chain as irregular spherical lumps. In anti-tumor activity assays, sulfation of PAS significantly improved the capacity to inhibit tumor cells growth by arresting the cell cycle progress in specific phases. It could be concluded that MW and molecular mass distribution had much greater influence on anti-tumor activities of SPAS against human non-small cell lung cancer A549 cells, human hepatocellular carcinoma HepG2 cells and human cervical cancer Hela cells. Microwave-assisted synthesis might possess widespread application in preparation of new polysaccharide derivatives with rich variety of structural features and bioactivities.