MicroRNAs, as non-coding RNAs, play important roles in many key physiological processes in organisms and can significantly impact gonadal development in fish through the regulation of target gene transcription. Therefore, this study investigated the previously identified key miRNA-novel_176 involved in gonadal development. Various experimental techniques including fluorescence quantitative PCR, fluorescence in situ hybridization, dual-luciferase reporter gene assay, RNA interference, and overexpression were employed to systematically validate its regulatory relationship with target genes and explore its potential biological functions. The results showed that miRnovel_176 was expressed in various tissues of Trachinotus blochii, with higher expression levels observed in the ovary than in the testis, although the difference was insignificant. miRnovel_176 was primarily localized in the cytoplasm of ovarian cells, with positive signals mainly detected in oocytes and granulosa cells. Weak hybridization signals were also observed in the supporting cells and spermatogonia of the testis. miRnovel_176 was found to target the 3'-UTRs of the esr2 and foxo3 genes, significantly inhibiting luciferase activity. Following transfection with novel_l76-antagomir and novel_l76-agomir, the expression levels of miRnovel_176 were significantly downregulated and upregulated, respectively, leading to significant opposite expression changes in the target genes foxo3 and esr2. This study indicates that the target genes of miRnovel_176 are esr2 and foxo3, and miRnovel_176 can regulate their expression levels by targeting their 3'UTR regions. This study provides new evidence for further revealing the function of miRNAs in the gonadal development of fish and also provides new candidate miRNAs for the study of gonadal development of T. blochii.