There are large differences in morphology and living habits of Japanese flounder () in the early stages of metamorphic development. These differences make an ideal model of metamorphosis in fish. The thyroid hormone (TH) plays a decisive role in the development of metamorphosis. The physiological role of TH is to regulate the transcription of target genes via the thyroid hormone receptors (TRs) and thyroid hormone response elements (TRE)-AGGT(C/A)A. In this study, was used to investigate a new method for finding the target genes regulated by TH. We expressed and purified the p3×Flag-TRαA and p3×Flag-TRβ fusion proteins to identify the target genes downstream of TH by using a cyclic amplification and selection of target (CAST) method. We found 11 potential TRαA target genes and 12 potential TRβ target genes. TRαA and TRβ share 4 identical potential target genes, which are ), aminoacyl tRNA synthetase complex interacting multifunctional protein 1 (). The expression of these genes was tested in different adult tissues and different metamorphosis development stages of in the NC (normal control), TH (thyroid hormone), and TU (thiourea) groups by real-time quantitative PCR. The expression of these genes was highest in the adult brain tissue (<0.05). The expression of these genes during early metamorphic development was highest at 17 dph (days post hatching) and lowest at 28 dph and 32 dph. The expression of these genes in the TH group was lower than in the NC group (<0.05). The difference between the NC group and the TH group at 17 dph was the largest (<0.05), while NC and TH groups had the smallest expression differences at 28 dph and 32 dph (>0.05). Compared to the TH group, the expression in the TU group increased slightly in each period (, and are target genes directly regulated by TH and that the expression of these genes is inhibited by TH. This study successfully identified an effective method to locate the target genes of TH. TH can inhibit the transcription of the target genes during metamorphosis. These results provide basic information for improving the thyroid hormone regulation network. However, further research is required to determine how TH inhibits the expression of these genes.