A newly isolated Japanese flounder (Paralichthys olivaceus) β-actin promoter and its derivative compact construct Poβ-actinΔ−1080/−801Δ−500/−201 have recently been demonstrated to promote ectopic gene expression in cell lines. Different Poβ-actin promoter deletion mutants were constructed and functionally characterized. Mutational analyses by dual-luciferase detected that three regulatory elements, including one enhancer (−1399/−1081) and two silencers (−1080/−801, −500/−201) in the first intron. The sequence located at −1399/−1081 was determined to significantly affect promoter activity. Additionally, the first exon (−1489/−1400) could also remarkably promote the β-actin promoter activity. In the following transduction application, we removed the two silencers and generated a compact reconstruct promoter/enhancer (Poβ-actinΔ−1080/−801Δ−500/−201), which exhibited relatively stronger promoter activity compared with Poβ-actin. Furthermore, the green fluorescent protein (GFP) transgenic stable flounder cell line was obtained by the reconstructed Poβ-actinΔ−1080/−801Δ−500/−201 promoter. Our study provided the potential application of Japanese flounder β-actin, particularly Poβ-actinΔ−1080/−801Δ−500/−201, in ectopic gene expression in the future.