The invention provides PLS3 recombinant protein eukaryotic expression plasmid as well as a construction method and an application thereof. The PLS3 recombinant protein eukaryotic expression plasmid carries a His labeled PLS3 recombinant protein coding gene, and the eukaryotic expression plasmid also carries a fluorescence reporter gene. By transfecting eukaryotic cells with the expression plasmid, an obtained cell line is applied to researches on such cell life functions as endocytosis, migration, invasion, chemotherapy drug resistance and the like. The eukaryotic expression plasmid is also applicable to extraction of eukaryotic Plastin-3 protein, screening of protein which mutually acts with the Plastin-3, screening of polypeptide participating in specific binding to the Plastin-3 and the like.