Conventional processing cannot fully modulate multiple biological activities of bioactive Polygonatum sibiricum polysaccharides using structural modifications and complex conformational transformations. The present study aims to clarify the different effects of steaming and fermentation—two representative processing techniques, including dynamic structural evolution of the medicinally valuable polysaccharides and the subsequent precise regulation of their bioactivities. Polysaccharides were extracted and then purified from fresh Polygonatum sibiricum, conventionally three-times-steamed Polygonatum sibiricum, and steamed-then-fermented Polygonatum sibiricum. The polysaccharide samples were designated as FPP, PPP, and FMP, respectively. An analytical platform was also employed: high-performance gel permeation chromatography (HPGPC) was used to determine molecular weight distributions; high-performance liquid chromatography (HPLC) with pre-column derivatization was used to quantify monosaccharide composition; Fourier transform infrared spectroscopy (FT-IR) was used to identify characteristic functional groups and conformational transitions. Zeta potential and dynamic light scattering analysis were used to assess colloidal stability and particle size uniformity, while scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to visualize morphological and nanostructure transformations, respectively. Antioxidant and hypoglycemic activities were evaluated through in vitro assays. Specifically, the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacities were determined with α-amylase and α-glucosidase inhibitory kinetics. Steaming was used to alter the structural integrity of the polysaccharides, disrupt their native triple-helix conformation, modify the monosaccharide profile, shift molecular weight distribution toward higher ranges, and dramatically increase uronic acid content. Subsequently, fermentation acted as the precise biological modification, further fine-tuning monosaccharide compositional ratios using microbial enzymatic hydrolysis. Biotransformation raised the absolute Zeta potential, electrostatic repulsion and colloidal stability, and particle size distribution, indicating the remarkable homogeneity. These conformations 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 generated a porous network morphology, thereby increasing structural porosity and specific surface area. According to these morphological shifts in AFM images, nanoscale transformations occurred from flexible, worm-like chains of FPP to compact, spherical chains in both PPP and FMP. The average chain height increased markedly after steaming and then decreased after fermentation. Functionally, these structural modifications were closely correlated with enhanced bioactivities. In antioxidant evaluations, PPP exhibited the most potent DPPH radical scavenging capacity. Meanwhile, FMP presented robust ABTS radical scavenging activity compared with the PPP. In hypoglycemic potential, both PPP and FMP were more effectively inhibited α-amylase than native FPP. Crucially, FMP shared the optimal inhibitory effect against α-glucosidase among all tested samples. Steaming and fermentation served as effective strategies to modulate the chemical structure, spatial conformation, and microscopic morphology of Polygonatum sibiricum polysaccharides, thereby enhancing their antioxidant and hypoglycemic activities. The processing-induced structural modifications showed a strong correlation between specific structural features and physiological functions in plant polysaccharides. Consequently, this finding can also provide a solid theoretical foundation and practical guidance for the precise, high-value industrial application of Polygonatum sibiricum resources in the functional food using traditional Chinese medicine processing.