Yeast plays an important role in fermented cereal products, especially fermented flour products, affecting the texture, aroma, appearance and nutrition of the products. In order to select grain microbial starter and improve the quality of fermented flour products, yeasts were isolated from traditional old flour and natural flour collected from different provinces and cities in China. Yeast identification was carried out by combining morphology and 26S rDNA sequence alignment. High-sugar-resistant, heat-resistant and high-quality yeasts were screened out to verify their application characteristics in steamed bread, toast bread and frozen dough. The results showed that 65 strains of yeasts were isolated, all of which were Saccharomyces cerevisiae, and 9 strains with strong fermentation ability were screened out, among which 5 strains could improve the specific volume, texture and taste of steamed bread, and the other 4 strains were resistant to high sugar and heat, which could improve the texture and flavor of toast bread. Besides, one strain had strong frost resistance, which could prolong the vitality period of frozen dough. The discovered strain is expected to be developed into a high-quality starter for fermented grain products, improve the performance of different kinds of fermented flour products, provide differentiated products for consumers, and promote the innovative development of grain processing industry.