The transcription factor oct4 plays an important role in maintaining the pluripotency of stem cells, early embryonic development and reprogramming of somatic cells. Construction of the eukaryotic expression vector of oct4 gene from Megalobrama amblycephala (Maoct4) can provide a practical tool for further study on the mechanism of the gene. The target gene fragment was obtained from the pCS2+Maoct4 vector containing the ORF sequence of the Maoct4 gene, and was cloned into pCVpr to construct the infusion and recombinant vector of pCMV-Maoct4-Red. After enzyme digestion and sequencing identification, the recombinant vector was transfected into HepG2 cells by liposome, and the expression and localization of the red fluorescence in HepG2 cells were observed in the nucleus, and the fusion protein was also detected by Western blot using MaOct4 antibody. Therefore, the fluorescent eukaryotic expression vector of pCMV-Maoct4-Red was successfully constructed, which could be effectively localized in the nucleus in eukaryotic cells,which would lay the foundation for further study on the function of the Maoct4 gene.