Abstract: A critically important and unresolved scientific issue is how traditional processing methods can significantly modulate the multiple biological activities of bioactive Polygonatum sibiricum polysaccharides through specific structural modifications and complex conformational transformations. To address this issue, the present rigorous study aims to systematically elucidate and compare the distinctive effects of steaming and fermentation — two representative traditional processing techniques. The focus is on the intricate dynamic structural evolution of these medicinally valuable polysaccharides and the subsequent precise regulation of their bioactivities. Polysaccharides were extracted and purified from fresh Polygonatum sibiricum, traditionally three-times-steamed Polygonatum sibiricum, and steamed-then-fermented Polygonatum sibiricum. The corresponding polysaccharide samples were designated as FPP, PPP, and FMP, respectively. A multi-technique analytical platform was employed for comprehensive characterization: high-performance gel permeation chromatography (HPGPC) determined molecular weight distributions; high-performance liquid chromatography (HPLC) with pre-column derivatization quantified monosaccharide composition; Fourier transform infrared spectroscopy (FT-IR) identified characteristic functional groups and characterized conformational transitions. Zeta potential and dynamic light scattering analyses assessed colloidal stability and particle size uniformity, while scanning electron microscopy (SEM) and atomic force microscopy (AFM) visualized morphological and nanostructural transformations, respectively. Antioxidant and hypoglycemic activities were evaluated through in vitro assays. These assays specifically determined the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacities, alongside α-amylase and α-glucosidase inhibitory kinetics. Steaming profoundly altered the structural integrity of the polysaccharides, disrupting their native triple-helix conformation, modifying the monosaccharide profile, shifting molecular weight distribution toward higher ranges, and dramatically increasing uronic acid content. Subsequent fermentation acted as a precise biological modification, further fine-tuning monosaccharide compositional ratios through specific microbial enzymatic hydrolysis. This biotransformation raised the absolute Zeta potential, enhanced electrostatic repulsion and colloidal stability, and drove particle size distribution toward remarkable homogeneity. These conformational changes were visually captured by microstructural observations. In SEM imagery, morphological transitions were observed: FPP displayed a smooth, continuous sheet-like film; steaming induced regularly arranged, protrusive structures; fermentation uniquely generated a highly porous network morphology, increasing structural porosity and specific surface area. Consistent with these morphological shifts, in AFM images, nanoscale transformations occurred from flexible, worm-like chains characteristic of FPP to compact, spherical chains in both PPP and FMP; the average chain height increased markedly after steaming and decreased after fermentation. Functionally, these structural modifications correlated directly with enhanced bioactivities. In antioxidant evaluations, PPP exhibited the most potent DPPH radical scavenging capacity. Meanwhile, FMP presented robust ABTS radical scavenging activity comparable to that of PPP. Concerning hypoglycemic potential, both PPP and FMP inhibited α-amylase significantly more effectively than native FPP. Crucially, FMP exerted the optimal inhibitory effect against α-glucosidase among all tested samples. Steaming and fermentation serve as highly effective strategies that synergistically modulate the intricate chemical structure, spatial conformation, and microscopic morphology of Polygonatum sibiricum polysaccharides, thereby significantly enhancing their antioxidant and hypoglycemic activities. These findings deepened the understanding of processing-induced structural modifications in plant polysaccharides and established a clear correlation between specific structural features and physiological functions. Consequently, this research provided a solid theoretical foundation alongside practical guidance for the precise, high-value industrial application of Polygonatum sibiricum resources in the functional food and nutraceutical sectors, offering new perspectives for the rational development of traditional Chinese medicine processing techniques.