The aim of this study was to investigate the regulatory effect of ddx27 gene on tp53 gene expression in zebrafish （Danio rerio） .The zebrafish ddx27 eukaryotic expression vector pCMV-3×Flag-ddx27 was constructed using homologous recombination technology based on the coding sequence （CDS） of ddx27 gene from NCBI online database.Sub-cellular localization and Western blotting were used to confirm the expression of ddx27 gene，the effect of ddx27 gene overexpression on tp53 gene expression was tested using dual-luciferase reporter gene assay.The results showed that the electrophoretic fragment size and sequencing alignment of ddx27 CDS region and positive clones were consistent with expectations.Western blotting showed that Ddx27 recombinant plasmid could be expressed normally，and the protein size was consistent with the predicted results.Sub-cellular localization showed that Ddx27 protein was expressed in the nucleus of HEK293T cells.Furthermore，the pCMV-3×Flag-ddx27 vector could significantly enhance the activity of tp53 promoter reporter gene pGL3-tp53-Luc，which was 1.8 times than that of the control group （P<0.05）.The above results showed that the zebrafish pCMV-3×Flag-ddx27 eukaryotic expression vector was successfully constructed and could be expressed in the mammalian nucleus，and ddx27 gene could promote the expression of tp53 gene.