Biodegradation based on microbial enzymes is considered to be one of the promising ways for controlling patulin contamination. However, few patulin degrading enzymes have been isolated and characterized until now. Here, a short-chain dehydrogenase/reductase (SDR) gene, CgSDR, was cloned from a yeast strain Candida guilliermondii, and expressed in Escherichia coli. The expression of CgSDR conferred a strong patulin tolerance and degradation ability to E. coli, and purified CgSDR could transform patulin into E-ascladiol in vitro with NADPH as a coenzyme. Moreover, addition of CgSDR at 150 μg/mL could reduce 80% of patulin in apple juice and the biodegradation process did not affect the quality of the apple juice. A molecular docking analysis and site-directed mutagenesis indicated that CgSDR might interact with patulin via VAL188 as an active binding sites. The findings provide new insights for developing enzymic formulations for mycotoxin detoxification in fruit derived products.