Tartary buckwheat is one of the world's minor cereal crops; it contains all 18 essential amino acids. Frozen baked goods made from raw materials such as tartary buckwheat have attracted increasing attention. Due to the strong carbohydrate metabolism function of Lactobacillus plantarum (Lp), the fermentation ability of Lactobacillus fermentum (Lf), and the ability of Weissella confuse (Wc) to produce a large amount of extracellular polysaccharides (EPSs) with certain anti-freeze properties during fermentation, the mixed fermentation of sourdough prepared by these strains will have a higher improvement effect on product quality. In this study, using HPLC, SDS-PAGE, and other methods, the growth of exogenous lactic acid bacteria in sourdough, major metabolic products (such as organic acids and sugars), and protein distribution, among other things, were investigated. The sourdough was then applied to produce frozen dough bread, and the quality characteristics of the bread products were characterized. The results showed that with the Lf+Wc mixed strain, the bacterial count reached 9.18 (lg(CFU/g))(based on the mass of sourdough) after 12 h of fermentation, with a large amount of fructose (11.82 mmol/kg) and EPS (3.132 g/kg) produced. Compared with other groups, it had a moderate acid production rate, lower proteinase activity, and a slower protein degradation rate. Furthermore, when the Lf+Wc group was applied to frozen dough bread, the degree of quality decline after 13 weeks of frozen storage was smaller, with a baking loss rate decreasing by only 5.3%. In addition, the GC-MS spectrometry analysis of flavor components found that after 13 weeks of frozen storage, there was an increase in eight volatile compounds (Lf+Wc frozen dough bread). In conclusion, Lf and Wc have good synergistic effects, with a fast sugar metabolism rate and the ability to produce a large amount of EPSs with anti-freeze properties, which also explains why the final frozen dough product has the best anti-freeze performance.