Abstract: To identify novel zearalenone （ZEN）‑degrading enzyme genes and achieve their heterologous expression in Escherichia coli， a previously isolated ZEN‑degrading strain， Bacillus velezensis L9， was employed as the source organism. First， differentially expressed genes under ZEN stress were analyzed by prokaryotic transcriptome sequencing， and coding sequences of potential degrading enzymes were screened through GO， KEGG， and COG functional annotations. Molecular docking was subsequently performed to simulate the interaction between the candidate proteins and ZEN. Second， using genomic DNA of B. velezensis L9 as a template， specific primers were designed for the candidate gene ctg1003， and the target fragment was amplified by PCR， recovered， and purified. The fragment was then ligated into the linearized vector pET‑32a（+） via homologous recombination to construct the recombinant expression plasmid pET‑32a‑ctg1003. After verification by double restriction enzyme digestion and sequencing， the confirmed recombinant plasmid was transformed into the expression host E.coli BL21（DE3）. Finally， the recombinant strain BL21（pET‑32a‑ctg1003） was induced with 1 mmol/L IPTG for 6 h， and the expression of the target protein was examined by SDS‑PAGE. Molecular docking suggested that the candidate gene ctg1003， encoding a putative hydrolase， possessed ZEN‑binding potential. A specific band of 1209 bp was successfully amplified from the L9 genome by PCR， and the sequencing result was consistent with the expected sequence. Following insertion of this fragment into the pET‑32a（+） vector， two fragments of 5896 bp and 1209 bp were obtained by double enzyme digestion， indicating that the recombinant expression plasmid was successfully constructed. After IPTG induction， a recombinant protein with a molecular weight of approximately 63.4 kDa （including the 6×His tag） was expressed in E. coli BL21（DE3） and was found to exist predominantly in a soluble form. In conclusion， the ZEN‑degradation‑related gene ctg1003 derived from B. velezensis L9 was successfully cloned， and its soluble expression was achieved in E. coli， thereby establishing a foundation for further investigation of the enzymatic properties and degradation mechanisms of this protein.