Searching for natural products with anti-tumor activity is an important aspect of cancer research. Seaweed polysaccharides from brown seaweed have shown promising anti-tumor activity; however, their structure, composition, and biological activity vary considerably, depending on many factors. In this study, 16 polysaccharide fractions were extracted and purified from three large brown seaweed species (Sargassum horneri, Scytosiphon lomentaria, and Undaria pinnatifida). The chemical composition analysis revealed that the polysaccharide fractions have varying molecular weights ranging from 8.889 to 729.67 kDa, and sulfate contents ranging from 0.50% to 10.77%. Additionally, they exhibit different monosaccharide compositions and secondary structures. Subsequently, their anti-tumor activity was compared against five tumor cell lines (A549, B16, HeLa, HepG2, and SH-SY5Y). The results showed that different fractions exhibited distinct anti-tumor properties against tumor cells. Flow cytometry and cytoplasmic fluorescence staining (Hoechst/AO staining) further confirmed that these effective fractions significantly induce tumor cell apoptosis without cytotoxicity. qRT-RCR results demonstrated that the polysaccharide fractions up-regulated the expression of Caspase-3, Caspase-8, Caspase-9, and Bax while down-regulating the expression of Bcl-2 and CDK-2. This study comprehensively compared the anti-tumor activity of polysaccharide fractions from large brown seaweed, providing valuable insights into the potent combinations of brown seaweed polysaccharides as anti-tumor agents.