Abstract: Phenylpropanoid aromatic substances, produced by biotransformation of trans-anethole, which are regarded as natural materials, were widely used in the food processing, flavour and fragrance producing and pharmaceutical industry. Pseudomonas sp. AT39 with the ability of degrading trans-anethole to anisaldehyde was used to construct mutants library including 6 137 mutants through inserting mutation by pTnMod-RKm’. 44 mutants of trans-anethole metabolism affected obviously were found. Compared to the wild-type strain, mutant AT39-15 displayed 14% of the wild-type strains in the ability of degrading the trans-anethole. Moreover, the results of hiTAIL-PCR and gene sequencing indicated that the size of gene was 1 047 bp which shared 100% identity with the trans-anethole oxidase gene（tao） from P. putida JYR-1.