To improve yeast survival under freezing conditions and reduce quality deterioration of frozen dough, the role of aromatic amino acid aminotransferase (Aro9) in yeast cryotolerance was investigated. An aro9-overexpressing strain (LKF-1-Aro9) and an aro9 knockout strain (LKF-1-ΔAro9) were constructed to verify the effect of Aro9 on the freezing tolerance of Saccharomyces cerevisiae. The survival rates of LKF-1 after 2, 5, 10, and 15 days of storage at −20 ℃ were 60.76%, 56.40%, 13.76%, and 8.63%, respectively, whereas those of LKF-1-Aro9 were 79.97%, 67.03%, 43.80%, and 29.30%, respectively. After 3 days of frozen storage, the times required for LKF-1, LKF-1-Aro9, and LKF-1-ΔAro9 to reach the stationary phase were 14, 12, and 21 h, respectively. Scanning electron microscopy revealed that LKF-1-Aro9 maintained a more intact cell surface structure after 7 days of storage at −20 ℃. Furthermore, after 3 days of storage at −20 ℃, the fluorescence intensities of LKF-1 and LKF-1-Aro9 after propidium iodide staining were 4 096.00 and 3 336.67, respectively, indicating greater cell membrane damage in LKF-1 than in LKF-1-Aro9. After storage at −20 ℃, the intracellular proline contents of LKF-1 and LKF-1-Aro9 were 0.35 and 2.19 μg•mg-1, respectively, suggesting that the higher proline accumulation in LKF-1-Aro9 conferred greater tolerance to freezing stress. Additionally, crystal violet staining showed OD595 values of 0.242 4 and 0.279 6 for LKF-1 and LKF-1-Aro9, respectively, indicating that LKF-1-Aro9 produced a thicker biofilm that better withstands adverse environmental conditions. In conclusion, overexpression of the aro9 gene effectively enhances freezing tolerance in S. cerevisiae. This improvement was closely associated with enhanced cell membrane integrity, increased biofilm formation, and elevated proline accumulation.