The coast of Zhejiang has the most extensive fishing grounds in China and is rich in fish resources. Juvenile Engraulis japonicus is one of the key fish species in the area. However, due to over fishing and other human factors, which resources are less than they used to be. The Ministry of Agriculture and Rural Affairs approved juvenile E. japonicus fishery along the coast of Zhejiang in 2018 to achieve a sustainable harvest of this species juvenile. Meanwhile, there were no studies report using DNA barcoding to identify species of the fishery presently. To comprehensively understand the bycatch species of juvenile anchovy fishery and investigate the effectiveness of mitochondrial cytochrome oxidase subunit I (CO Ⅰ) gene in the identification of fish, this study adopted the method of DNA barcode-assisted morphological classification, taking 192 juvenile fishes from the by-catch of juvenile E. japonicus fishery. Finally, we identified 48 species, belonging to 46 genera of 2 classes 11 orders 36 families, of which 46 species were identified to the level of species, 2 to the level of genera, and 11 species of morphology misidentifies were successfully identified. The average length of 152 CO I sequences was 654 bp (634-656 bp), and conserved sites accounted for 47.21%, informative sites accounted for 45.25% and variable sites accounted for 51.89%. The content of C+G (47.66%) was significantly lower than that of A+T (52.34%), showing at AT bias, and the highest AT content was found at the site 3. The average interspecific genetic distance (0.252 3) was 74 times higher than the average intraspecific genetic distance (0.003 4). The largest genetic distance between genera was between Salanx and Pampus (0.339 5), and the smallest was between Trachurus and Alectis (0.119 7). The largest genetic distance between the families was between Salangidae and Stromateidae (0.339 5), and the smallest was between Atherinidae and Carangidae (0.192 1). The largest genetic distance was between Perciformes and Osmeriformes (0.276 2), and the smallest was between Scorpaeniformes and Atheriniformes (0.212 2). The ABGD analysis resulted in 48 OTUs, consistent with the morphological classification. The phylogenetic tree was constructed based on CO Ⅰ gene, and individuals of the same species could be clustered in the same branch. The species could be effectively distinguished, but the clustering relationships at the family and order levels were not clear. The DNA barcoding identification results were basically consistent with the morphological identification results. The above results indicated that DNA barcoding could be used as an effective method of identification of juvenile E. japonicus fishery in Zhejiang coast, which made up for the limitations and shortcomings of traditional morphological identification methods. The findings of this study can lay the foundation for the study of fish diversity and spawning migration along the Zhejiang coast, as well as provide a scientific basis for the sustainable development and utilization of juvenile Engraulis japonicus fishery.