One fundamental question in biology is how animals control their proper body size. Hippo signaling pathway has emerged as an evolutionarily conserved network to regulate organ size from Drosophila to mammals. Interestingly, our previous study has also implied the roles of hippo signaling in Chinese tongue sole (Cynoglossus semilaevis), a flatfish exhibiting female-biased sexual size dimorphism (SSD). Specifically, two yes-associated protein 1 (yap1) homologs, key effectors within hippo signaling, exhibited the opposite expression pattern in the female and male gonads of C. semilaevis. Whether and how these two yap1 genes participate in female-biased SSD is unknown. In the present study, phylogenetic tree analysis firstly designated these two yap1 genes as yap1 and yap1b, with no taz gene available in C. semilaevis. Subsequent quantitative PCR analysis demonstrated that yap1 exhibited the highest expression levels within the male gonad at three-month-old, while the predominant expression of yap1b was detected in the female gonad at two-year-old. The co-localization of yap1 and yap1b was observed in the cytoplasm of oocyte and sperm. Cebpα, a negative regulator for dmrt1, exhibited positive and negative regulation on the yap1 and yap1b, respectively. Myog, a crucial transcription factor for myogenesis, specifically binded and activated yap1b promoter, not yap1. Furthermore, DAP-seq revealed the regulatory networks of yap1 and yap1b in C. semilaevis. Importantly, many growth and reproduction-related pathways, including the JAK-STAT signaling pathway, prolactin signaling pathway, axonal regeneration, and steroid hormone synthesis, were included in the common peaks of yap1 and yap1b. Moreover, the application of verteporfin reagents into testis and ovary cells resulted in a decrease in yap1 and yap1b levels. Consequently, the expression of growth hormone receptor (ghr), growth hormone actin α (smtla), estrogen receptor (esr2b), and 17β-hydroxysteroid dehydrogenase type 7 (hsd17b7), were also affected. In addition, the administration of verteporfin on C. semilaevis caused a significant reduction in growth rate. These findings provided insights into the role of the hippo signaling pathway in regulating SSD of C. semilaevis. •Yap1 and yap1b were identified in C. semilaevis, with no taz gene.•The co-localization of yap1 and yap1b was observed in the cytoplasm of oocyte and sperm.•Cebpα, a negative regulator for dmrt1, showed positive and negative regulation on the yap1 and yap1b, respectively.•Dap-seq revealed that yap1 and yap1b regulated JAK-STAT, prolactin, and steroid signaling pathways.•Verteporfin treatment caused a decrease of yap1/yap1b and a reduction in C. semilaevis growth rate.