Abstract: As one of the main strains of fermented milk, Streptococcus thermophilus can impart excellent texture, rich nutritional value and unique flavor to fermented milk during the fermentation and storage process of fermented milk. On the basis of the previous research, 7 strains of S. thermophilus with good fermentation characteristics isolated from traditional fermented milk in different regions were used as the experimental strains this experiment, and commercial starters were used as the control group, solid phase microextraction was used combined with gas chromatography-mass spectrometry technology to detect and analyze the volatile flavor substances in the fermented milk at the end of the fermentation, which was pH4.5. The results showed that among all the experimental strains, the composition and content of volatile flavor compounds in G80-2 fermented milk were closest to the control group. On this basis, the dynamic analysis of the volatile flavor substances produced by the G80-2 strain during the fermentation and storage of milk was carried out. It was found that the strain was detected acids (4 types), alcohols (10 types), ketones (12 types), aldehydes (6 types), esters (1 type) and other compounds during fermentation and storage. Some main characteristic flavor substances such as acetic acid, acetaldehyde, diacetyl, acetoin, 2-heptanone, 1-heptanol, etc. were relatively high in content. It was concluded that the strain could be used as a starter in the production of dairy products.