Abstract: In this study, the home-made grape wine was used to study the selective degradation behavior of hexaconazole enantiomers and their effects on the growth of Saccharomyces cerevisiae during wine processing. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the residue levels of hexaconazole enantiomers in wine samples at different fermentation stages, and the quantity of Saccharomyces cerevisiae in wine samples were detected by flat colony counting method. The results showed that the average recoveries of hexaconazole were in the range of 79%-111% with the relative standard deviations (RSD) of 4%-28% for the established method. A gradual decrease in hexaconazole residues was observed during the wine process, and no significant differences were found in degradation behavior of the two enantiomers. The degradation rates of (+)-hexaconazole and (−)-hexaconazole were 41.2% and 45.9% with half-lives of 13.97 and 12.38 d, respectively. The contents of Saccharomyces cerevisiae in hexaconazole-treated samples significantly decreased compared to the control group, and the quantity ratio reached a maximum of 2.73∶1 at 8 days after the treatment. In summary, wine processing effectively reduced the residues of hexaconazole while it inhibited the growth of Saccharomyces cerevisiae. The results of this study provide a scientific basis for evaluating the health risk of hexaconazole during wine processing and its effect on wine quality.