Abstract: This study investigated the synergistic fermentation of whole wheat sourdough using Lactobacillus curvatus (Lc-39) and Wickerhamomyces anomalus (Wa-5). The systematic evaluation of fermentation characteristics was conducted by means of dynamic monitoring of microbial proliferation, pH, total titratable acidity (TTA), and nutritional components. The optimal freeze-drying protectant was screened based on bacterial survival rates and acid-producing capacity. Results demonstrated that synergistic fermentation with an initial inoculum ratio of Lc-39 to Wa-5 (L:Y=5:1) and Wa-5 at 10⁷ CFU/g achieved optimal fermentation activity. The matured sourdough exhibited superior physicochemical properties including a pH of 3.82 and total titratable acidity (TTA) of 13.41 g/kg. Notably, this treatment yielded the lowest amylose/amylopectin ratio (19.46%) alongside maximized functional properties: total antioxidant activity (0.34 μmol/g), flavonoid content (2.69 mg/g), total phenolic content (3.60 mg/g), amino acid concentration (911.72 μmol/g), and reducing sugar content (50.60 mg/g). Additionally, it achieved exceptional phytate degradation efficiency (98.37%), indicating enhanced nutrient bioavailability. The addition of 15% trehalose effectively preserved the viable bacterial count in whole wheat sourdough during freeze-drying, with survival rates of 83.38% for Lc-39 and 88.29% for Wa-5 strains. Following 90 days of ambient storage, there was no statistically significant decrease in the acid-producing capacity and acidification rate of the freeze-dried sourdough starter. Whole wheat bread fermented with this freeze-dried sourdough starter culture achieved the highest overall sensory rating of 7.8 in comparative assessments, demonstrating superior organoleptic properties. This study provides critical technical fundamentals and microbial strain resources essential for advancing the development of highly active whole wheat sourdough starters.