In this study, the influences of ultrasonic treatments with different working modes (fixed frequency and sweeping frequency) and various frequencies on the bacterial growth and metabolism yield of Saccharomyces cerevisiae (S. cerevisiae) were investigated by employing an in situ ultrasonic irradiation slot coupled with a flask fermentation equipment. The results revealed that the in situ ultrasonic treatment could promote the bacterial growth and metabolism yield, and the effect of fixed frequency ultrasound (FF) was higher than sweeping frequency ultrasound (SF). The content of metabolite in the fermentation broth increased with FF and decreased after SF treatments. On the other hand, when the frequency of FF was more than 33 kHz, the growth of S. cerevisiae became weaken, and the mortality rate of S. cerevisiae increased in fermentation broth. At FF of 23 kHz and 48 h fermentation-time, ethanol content increased by 19.33%, and the content of β-phenylethanol and other volatile metabolites such as esters also increased. In conclusion, FF could significantly improve the growth of S. cerevisiae, and the FF of 23 kHz showed the optimum impact on fermentation process of S. cerevisiae.