Abstract: To identify target genes mediated by thyroid hormone/thyroid hormone receptor TRαA in Paralichthys olivaceus, the CDS region of TRαA gene was cloned by RT-PCR and constructed a recombinant eukaryotic expression vector p3×Flag-TRαA. The recombinant plasmid was transfected into HEK293T cells, and the results demonstrated that the TRαA of Paralichthys olivaceus was successfully expressed in the mammalian expression system. Cell lysates transfected with recombinant plasmid were purified and filtered by Flag affinity chromatography to obtain the pure fusion protein TRαA. The dual luciferase reporter assay was performed in HEK293T cells that p3×Flag-TRαA was co-transfected with the reporter gene expression vector pGL3-Pro-atoh8-1517/1333/708 containing the candidate target promoter. The results support that the receptor of TRαA binds to two TRE recognition sequence specific to the–1497— –688 promoter region of the atoh8 gene to initiate the gene transcription, and that atoh8 is a target gene directly mediated by thyroid hormone via TRαA. This study provides the basis for further exploration of thyroid hormone-regulated thyroid hormone receptor TRαA-mediated signaling pathways.