Abstract: In this study we selected 313 bp sequence of cytochrome C oxidase subunit Ⅰ (COⅠ) gene as miniDNA barcode to evaluate the applicability of barcode in the identification of fishes at the species level. A total of 355 barcode sequences were obtained from 85 species in 66 genera of 38 families and 11 orders of fishes in Taiwan Strait. The average contents of T, C, A and G were 29.50%, 30.10%, 24.90% and 15.50%, respectively. The AT content was higher than 50% in each species. The average Kimura two parameter (K2P) distances within species, genera, families, orders and classes were 0.37%, 18.10%, 22.10%, 25.40% and 27.80%, respectively. The mean interspecific distance was 49-fold higher than the mean intraspecific distance. The K2P neighborjoining trees based on the sequence generally clustered species in accordance with their taxonomic classifications. High efficiency of species identification was demonstrated in the present study by miniDNA barcoding. Thus, we conclude that miniDNA barcoding can be used for the classification of fishes.