Abstract: To investigate the preparation of freeze-dried Pediococcus pentosaceus sourdough and its effects on the quality of multigrain steamed bread, this study utilized skim milk powder as a cryoprotectant to prepare freeze-dried P. pentosaceus sourdough. The cryoprotective effects of different skim milk powder concentrations were evaluated, and the ability of the freeze-dried sourdough to improve the textural quality of sorghum steamed bread was further explored. Results indicated that the survival rate of P. pentosaceus in freeze-dried sourdough with 10% skim milk powder was 28.5%, significantly lower than those with 15% and 20% skim milk powder. However, this group exhibited the highest dough fermentation capacity, a specific volume of steamed bread (2.58 mL/g), and sensory scores. An in-depth analysis of macromolecular structures revealed that sourdough fermented with 10% skim milk powder showed a significant decrease in β-sheet content and an increase in α-helix content in gluten protein secondary structures, alongside a notable rise in starch gelatinization temperature and a reduction in enthalpy. Applying the 10% skim milk powder freeze-dried P. pentosaceus sourdough in sorghum steamed bread preparation significantly improved the dough's textural properties. It enhanced the overall sensory score of the steamed bread. This study provides insights into the commercial production of freeze-dried P. pentosaceus sourdough and its application in multigrain steamed bread.