Fish comprise over 50% of the total number of vertebrates globally, with an estimated 60,000 species, of which more than 35,000 species have been recorded. The creation of a taxonomically exhaustive and comprehensive reference sequence database for fish is of utmost importance to facilitate the widespread implementation of DNA barcoding and metabarcoding methodologies in the monitoring and evaluation of aquatic ecosystems. A total of 21,535 COI barcodes and 1,074 18S rRNA barcodes of 21,589 fish species were provided, belonging to 8 classes and 90 orders.