ABSTRACT Polysaccharides are natural polymers that widely exist in medicinal plants. Polysaccharides are complex molecules composed of long chains of monosaccharide units joined by glycosidic bonds from a structural point of view. Modern pharmacological research shows that the molecular weight (MW) of polysaccharides is closely related to their bioactivity. This review summarizes the preparation and detection methods of medicinal plant polysaccharides with different molecular weights. Subsequently, the paper analyzed the general experience of the correlation between bioactivity and their relative MW of polysaccharides from medicinal plants. The results showed that the polysaccharides with large relative MW (> 100 kDa) play a leading role in immune regulation. Polysaccharides with medium relative MW (10–100 kDa) play a leading role in the protection of the liver. Polysaccharides with small relative MW (< 10 kDa) play a leading role in the antioxidant effect, regulation of intestinal flora, and regulation of blood glucose. Consequently, the precise development of polysaccharides from medicinal plant resources based on relative MW is expected to be a shortcut for searching high biological activities of polysaccharides and provide a theoretical reference for polysaccharide research.