Spermatogenesis is the process during which spermatocytes undergo meiosis to produce spermatozoa. Sertoli-Sertoli and Sertoli-spermatogenic junctions are important for the maturity and release of sperms. These junctions, which are related to male infertility, have been reported in mammals but are unclear in teleost species. In this study, the RNAs in the gonads of 1.5-year-old Japanese flounders were sequenced using Illumina. The miRNA expression profiles in the mature gonads of males and females were characterized. A total of 277 mature miRNAs were screened in both gonads. Among them, 25 miRNAs were expressed differently in the ovary and testis. Afterward, miR-1388 was selected for its high expression level in the ovary. Gene ontology analysis revealed that the target genes of miR-1388 were highly involved in cell-cell adhesion. Nectin2l, the target of miR-1388, is a Ca-independent cell adhesion molecule that was demonstrated to be testis-biased via quantitative real-time PCR and located in Sertoli cells and spermatocytes via in situ hybridization. Our present study predicted the potential functions of sex-biased miRNA and indicated that miR-1388 regulates cell junctions by inhibiting nectin2l, which may have an important role in the later development of testes. Our study lays the ground work of miRNAs' function in the cell junctions of germ cells.