The Japanese flounder () is an ideal model for studying the metamorphosis of fish. Thyroid hormone (TH) can regulate the right eye shift and lifestyle of as it transforms from a bilateral pelagic larva into an asymmetrical benthic juvenile. At the same time, microRNAs (miRNAs) play a key role during metamorphosis. To research the regulation mechanism of TH, pol-miR-124, and Otx2 in , based on its binding site through bioinformatics analysis. First, we detected the expression levels of pol-miR-124 and Otx2 in adult tissues in the normal group and TH treatment group larvae using real-time quantitative PCR. Then, the 400 bp alignment region of Otx2 3 UTR containing the "seed sequence" was cloned, and the target relationships between miR-124 and Otx2 were verified at the cellular level by constructing a dual luciferase reporter. The qPCR results showed that both pol-miR-124 and Otx2 were highly expressed in the brain and eye tissues; the highest expression at 28 dph was observed in the larvae. Compared with the NC group, the pol-miR-124 expression level was inhibited at 20 dph and 24 dph in the TH group, and the Otx2 expression level was higher than that in the NC group. However, the expression level of pol-miR-124 increased at 28 dph, 32 dph, and 36 dph, but the Otx2 level was lower than that in the NC group. The expression of pol-miR-124 was negatively correlated with the expression of the predicted target expression. Luciferase results showed that the luciferase activity of the pol-miR-124 group was significantly decreased and was the target gene of pol-miR-124. The results of this study form an important research foundation for further revealing the developmental mechanism of the photoreceptor system during metamorphosis.