The albinism in Japanese flounder aquaculture has become a common phenomenon and influenced its large-scale farming and market value. Based on the albinism and normal Japanese flounder transcriptome and miRNA sequencing, tyrosinase related protein 1(tyrp1) and mmu-miR-143-5p_R+2 (mmu-143) were chosen for expression pattern, target gene prediction and verification analysis. Firstly, tyrp1a and tyrp1b were screened and identified by RACE and phylogenetic analysis. Subsequently, RNA hybrid was used for the prediction of the targeting relationship between tyrp1a and miRNA mmu-143, which was further verified by dual luciferase experiment. Finally, the results of the quantitative real time-PCR (qRT-PCR) showed that the expression of tyrp1a gene in normal skin of Japanese flounder was significantly higher than the albinism skin, and the expression of mmu-143 in normal skin was significantly lower than the albinism skin. The present study identified two transcripts of tyrp1a and tyrp1b from Japanese flounder. Dual luciferase experiment and qRT-PCR analysis confirmed that tyrp1a was the target gene of mmu-143, and mmu-143 affected the albino of Japanese flounder by regulating tyrp1a. The results of this study will be helpful to fully understand the molecular mechanism of Japanese flounder albinism.