Patulin is a common contaminant that frequently occurs in apple fruits and their derived products and constitutes a serious threat to human health. In the present work, the yeast strain Pichia guilliermondii S15-8, with high patulin degradation ability, was screened from nine probiotic yeasts. Subsequently, the optimum degradation conditions were finally established at 30 °C in 5 mL of juice simulated solution with 1.0 mg/L initial patulin for 24 h of degradation, and more than 90% of patulin could be degraded with a cell density of 1 × 10 8 cells/mL. Further investigation of the patulin stress response indicated that Pichia guilliermondii S15-8 had a strong tolerance to high patulin stress, even up to 100 mg/L. Furthermore, the product of intracellular enzyme degradation of patulin was identified as E-ascladiol through HPLC and LC–MS. These results corroborated the potential of probiotic Pichia guilliermondii S15-8 for patulin detoxification of apple-derived products. • Excellent degradation strain screening and degradation condition establishment. • High patulin stress tolerance of P. guilliermondii S15-8. • E-ascladiol was identified as patulin degradation product.