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Cell transfection is a specialized technique for introducing exogenous molecules, such as RNA and DNA, into cells and has become a prevalent method in gene functionality studies. The most frequently employed cell transfection methods include electroporation, viral infection, and liposome transfection. Liposome transfection is a prevalent technique in molecular biology for studying gene function and regulation. Liposome transfection has the advantages of wide applicability, superior efficiency, and simple operation. However, most liposome transfection reagents on the market are designed based on mammalian cells. Cell membrane composition, temperature adaptation range, and oxygen demand in teleost cells differ substantially from those in mammalian cells. Accordingly, the efficiency of liposome transfection in teleost cells is limited, and obtaining stable transfected cells directly through screening is challenging. Therefore, after the transfection of conventional transient expression vectors into teleost cells via liposomes, most of the plasmid copies tend to vanish due to cell proliferation after the duration of screening. Furthermore, the insignificant number of cells retaining plasmids makes it difficult to correctly integrate plasmids into the genome sequence, which hinders screening for stable cell lines. In this study, we investigated a method of rapidly obtaining stable transfected cells of Japanese flounder by transfecting the Tol2 transposon recombinant vector with liposomes. The CMV-EGFP-pminiTol2 vector was modified from the pminiTol2 plasmid, sustaining the functionality of the Tol2 transposable element to facilitate the highly efficient insertion of target genes into genomic DNA. The CMV-EGFP-pminiTol2 plasmid was highly suitable for stable transformation screening in Japanese flounder cells. This advantageous trait was mainly attributed to the following four aspects: (1) The CMV-EGFP-pminiTol2 plasmid carries autonomous transposon elements that can significantly enhance the integration ability of target genes. (2) The CMV-EGFP-pminiTol2 plasmid carries EGFP, allowing researchers to visualize transfection and screening effects directly via fluorescence microscopy. (3) The CMV-EGFP-pminiTol2 plasmid incorporates a neomycin resistance selection marker, which can be used to quickly and successfully screen transfected cells after G418 screening. (4) The multiple cloning site (MCS) region of the CMV-EGFP-pminiTol2 plasmid is mutated to retain two unique cleavage sites (XhoI and XmaI); plasmids can be linearized through these two sites, allowing the target gene to be inserted using homologous recombinase cloning. CMV-EGFP-pminiTol2 and pCS-TP plasmids were co-transfected into flounder cells via liposome encapsulation, and stable transfected cells were obtained through neomycin (800 μg/mL) screening for 2–3 weeks. After 2 weeks of screening, cells were stably cultured for 2–3 weeks without G418 screening. Finally, the EGFP fluorescence of the cells was observed using an inverted fluorescence microscope to confirm the transfection efficiency of the cells. The results revealed that the CMV-EGFP-pminiTol2 plasmid-transfected experimental group successfully expressed EGFP in all cells after G418 selection. In the pEGFP-C1- or pEGFP-N1-transfected control group, only a minor fraction of cells (≤10%) sustained EGFP expression after G418 screening. The CMV-EGFP- pminiTol2 vector is therefore suitable for the preparation of stable transgenic cells in Japanese flounder, offering numerous advantages, including reduced screening time, stable exogenous gene expression, simplified operations, and relatively low cost. Consequently, researchers can insert their desired target genes into the CMV-EGFP-pminiTol2 vector to construct stable transformed cells for investigations such as functional analysis and assessments of subcellular localization and long-term regulation. This study describes a novel approach to rapidly screening stably transformed flounder cells using the Tol2 transposon system. The results can be applied to molecular biology research in flounder and provide novel insights for constructing and screening stable transformations in teleost cells.