Abstract: 【Objective】This work was aimed to establish a identification method of Aeromonas based on 16S rRNA sequence and gyrB gene tandem DNA characteristic sequence， and to provide research technical support for the differentiation of Aeromonas at the species level.【Method】 The aquatic bacteria，which were detected as Aeromonas later，isolated from Fujian，Jiangsu and other places in China from 2020 to 2021 and ten different species of Aeromonas that have completed genome sequencing on NCBI were taken as the experimental objects. The 16S rRNA sequence，gyrB gene and the new DNA characteristic sequences obtained from the tandem of the two sequences were used as the basis for the establishment of the evolutionary tree. The results were identified by comparing different evolutionary trees，meanwhile，exercise tests，hemolysis tests，and sugar fermentation tests were also be used for auxiliary identification.【Result】The results showed that the combined tree had better discrimination effect than the single gene tree. In the phylogenetic evolution tree constructed based on single 16S rRNA sequences，A. media，A. hydrophila，A. dhakensis，and A. enteropelogenes were dispersed on different branches，and did not form independent species and evolutionary branches. In the phylogenetic evolution tree constructed based on the single gyrB gene，A. dhakensis，A. caviae，and A. hydrophilawere clustered on the same branch，while A. allosaccharophila and A. veronii were also clustered on the same branch. By combining 16S rRNA sequence and gyrB gene in tandem to form a new DNA characteristic sequence，the phylogenetic evolutionary tree was constructed，which could completely and effectively isolate different strains of Aeromonas，and cluster different species of Aeromonas into an independent branch，so as to achieve better interspecific differentiation.【Conclusion】 The phylogenetic tree constructed by combining 16S rRNA sequence and gyrB gene in tandem to form a new DNA sequence has better identification ability than the phylogenetic tree constructed solely based on 16S rRNA sequence or gyrB gene. It is an ideal method for genus species identification with high conserved sequence similarity. Therefore，in the case of the identification of Aeromonas by 16S rRNA sequencing，gyrB gene can be used as the sequencing object again，and after obtaining sequence information，the phylogenetic evolutionary tree can be constructed in tandem with 16S rRNA sequence to more accurately identify Aeromonas to the species level.