In this study, three polysaccharides (RHC, RHH, and RHA) were sequentially extracted from Rheum officinale Baill., under different conditions, and their sulfated, phosphorylated, and selenated derivatives were successfully synthesized. Results indicated that the sequential extraction method effectively separated polysaccharides with distinct structures. According to their charge properties, these three polysaccharides were identified as acidic polysaccharides with different structural characteristics. Activity assays showed that the phosphorylation modification of polysaccharides significantly enhanced the scavenging of hydroxyl, superoxide, and DPPH radicals, as well as the inhibitory effects on HepG2 and HeLa cells. Phosphorylated derivatives also promoted proliferation, phagocytosis, and nitric oxide (NO) release in RAW264.7 macrophages. In the 50-800 µg/mL range, these polysaccharides and their derivatives exhibited no cytotoxicity to RAW264.7 cells and significantly promoted cell proliferation, phagocytosis, and NO release. Notably, RHC and its derivatives have the most prominent antitumor and immunomodulatory effects. Overall, R. officinale polysaccharides and their derivatives, especially phosphorylated ones, have potential applications in health food and pharmaceuticals.