Zearalenone (ZEN) is a harmful macrolide mycotoxin, posing a serious hazard to human health. In this study, a highly efficient ZEN-degrading bacterium Gordonia hydrophobica HAU421 was isolated from soil by using spiramycin (SPM)-containing selective medium. Mass spectrometry analysis revealed that strain HAU421 could transform ZEN into hydrolyzed zearalenone (HZEN), zearalenol (ZEL), and hydrolyzed zearalenol (HZEL). A novel lactonohydrolase GhZH capable of hydrolyzing ZEN was mined from the genome of strain HAU421 and heterologously expressed in Escherichia coli. The recombinant GhZH exhibited peak activity at pH 7.0 and 42 °C. The catalytic triad of GhZH was identified as S122-D147-H297 via sequence comparison, molecular docking and site-directed mutagenesis. Moreover, toxicological analysis suggested that GhZH-catalyzed ZEN hydrolyzation resulted in the detoxification of its hepatotoxicity. To meet the industrial demands, GhZH was immobilized onto chitosan microspheres using the crosslinker glutaraldehyde. The stability of immobilized GhZH at harsh acidic pH and high temperature was enhanced in comparison with free GhZH. The immobilized GhZH achieved a ZEN removal rate of 53.2 % in beer and 74.0 % in corn steep liquor. These findings offer new insights into microbial ZEN degradation and support the advancement of enzyme-catalyzed ZEN detoxification.