Imazalil is a chiral fungicide widely used to protect strawberries against gray mold, which may pose threats to food safety. This study aims to investigate the stereoselective behavior of imazalil during strawberry growth and strawberry wine production. A method was proposed and validated for the extraction and quantitative analysis of imazalil residues in strawberry, strawberry pomace, and strawberry wine by using ultra-high performance liquid chromatography-tandem mass spectrometry. The method exhibited mean recoveries ranging from 86.2% to 119.7% with relative standard deviations of 0.1–11.3%. The dissipation curve of imazalil during strawberry growth followed the first-order kinetic model with a half-life ranging from 6.5 to 7.1 days. Significant enantioselectivity of imazalil was observed in strawberry grown under field conditions and strawberry wine production process, with enantiomeric fraction values ranging from 0.51 (2 h) to 0.42 (27d) and from 0.48 (0d) to 0.52 (10d), respectively. (+)-imazalil was preferentially degraded in strawberry under field conditions, while (−)-imazalil was preferentially degraded during the fermentation process. The processing factor was lower than 1 for each procedure, indicating that the wine-making process can reduce imazalil residue in strawberry. These findings may facilitate a more accurate risk assessment of imazalil and provide important guidance for the safe and efficacious use of imazalil in agriculture.