The ubiquitin-proteasome system is an important protein modification pathway, involved in many biological processes. Ubiquitin-conjugating enzyme E2 is the key enzyme in regulating protein ubiquitination. In order to explore the function of ubiquitin-conjugating enzyme E2 in female gonad development of Cynoglossus semilaevis, the Open Reading Frame (ORF) of Cs-UBE2D4 gene was cloned in this study. Its expression pattern was measured in various tissues of male and female adults, and at different developmental stages of gonads as well. Then, we constructed Cs-UBE2D4 promoter reporter vector (pGL3-Cs-UBE2D4) and detected its activity. The ORF of Cs-UBE2D4 gene was 360 bp, encoding 119 amino acids with a conserved ubiquitin-conjugating enzyme E2 domain. qPCR results showed that Cs-UBE2D4 was widely expressed in all tissues of females, especially in ovary and liver, but it was hardly detected in males. The gene expression increased from 90 days post-hatcing (dph), peaked at 6 months post-hatcing (mph), and decreased afterwards. Its expression level was lowest at 1.5 years post-hatching (yph) and increased at 3 yph. During this period, its expression was 1.97-fold higher at 6 mph than that at 90 dph. Dual luciferase assay showed strong activity of the promoter of Cs-UBE2D4 gene, indicating its participation in transcription. This study provides a theoretical basis for future research on Cs-UBE2D4 gene in ovarian development and gender-related mechanisms of Cynoglossus semilaevis.