Fastq files of MiFish and MiFish-tuna libraries constructed from extracted DNA in fish meal (FM) from urban fisheries biomass (UFB) in Japan collected from October 2020 to October 2021, and FM which was made from muscle tissues of yellowtail (Seriola quinqueradiata), pacific blue fin tuna (Thunnus orientalis) and yellowfin tuna (Thunnus albacares) for a validation study. The libraries were sequenced using the MiSeq system and Miseq Reagent Kit v3 (Illumina, San Diego, CA, USA) on 2 Ã? 300 bp read lengths. The purpose of this study is to show that the DNA metabarcoding method can contribute to improve traceability of FM from UFB in Japan.