The variations in melatonin and its isomer content were studied during ethanol fermentation by three different Saccharomyces cerevisiae strains, that is, A, B, and F. Additionally, the relationship between melatonin/melatonin isomer and the fermentation process was preliminarily explored and verified by wine fermentation of four types of berries (strawberry, blueberry, mulberry, and raspberry). The results showed that the melatonin content reached the maximum on day 1 and then decreased rapidly. Maximum values obtained for S. cerevisiae strains A, B, and F were 0.773, 0.647, and 0.825 ng/mL, respectively. The content of melatonin isomer reached the maximum on day 3 and then gradually decreased toward later stages. The maximum values obtained for S. cerevisiae strains A, B, and F were 34.89, 19.24, and 26.79 ng/mL, respectively. Thus, the content of the melatonin isomer was significantly higher than that of melatonin (p < 0.01). However, there were no significant differences in melatonin content before and after wine fermentation of the berries (strawberry, blueberry, mulberry, and raspberry), whereas the melatonin isomer content after fermentation was 64.4, 7.37, 81.9, and 55.5 ng/mL, respectively, which was significantly higher than the melatonin content. In summary, melatonin and its isomer were produced in the early fermentation stage and their individual content gradually decreased during the mid-to-late stage, which was speculated to be closely related to the stress response by S. cerevisiae during early stages of fermentation.