The invention relates to a construction method and applications of engineering strain for delivering mammalian cell protein. The construction method comprises the following steps: deleting four virulence factors including exoS, exoT, exoY and ndk genes from pseudomonas aeruginosa PAK strain genome, so as to have no cytotoxicity to mammalian cells; further deleting popN gene, for inhibiting bacterial III type secretory system (T3SS), from chromosome, so as to remarkably increase the injection quantity of T3SS-mediated protein, thus constructing the engineering strain delta5. The constructed engineering strain delta5 can be used for delivering proteins of various mammalian cell linings, performing gene editing, cell reprogramming, protein function research and other work. The types of delivered cells are wide, including human and mice skin cells, muscle cells, intestinal cells, liver cells, multipotent stem cells and the like.