Objective To express the humanized immunoapoptotic protein with a modified cleavage site in mammalian cell expression system and verify its killing effect on human epidermal growth factor receptor 2 (HER2)-positve tumor cells. Methods Kozak sequence, the humanized anti-HER2 single chain antibody P1h3, Fc tag, cathepsin D cleavage site D1 and pro-apoptotic effector tBid gene were linked in sequence and cloned into the eukaryotic expression vector pLVX-EGFP. Cell culture supernatant was harvested and subjected to Western blot analysis to detect the secretory expression of the recombinant plasmid after its transfection into HEK293F cells. Killing effect of the recombinant plasmid on HER2-positive PC9 tumor cells was evaluated by cell counting and luciferase activity assay. Results The eukaryotic expression vector pLVX-P1h3-Fc-D1-tBid-EGFP was successfully constructed and its expression was confirmed by Western blot analysis. The humanized immunoapoptotic proteins significantly induced the death of HER2-positive PC9 cells. Conclusion The modified humanized immunoapoptotic proteins obtained by mammalian cell expression system could specifically kill HER2-positive tumor cells.