The invention belongs to the field of biotechnology, and relates to a slightly acidic environment targeted polypeptide modified tumor targeted nano drug delivery system, and a preparation method thereof. The slightly acidic environment targeted polypeptide modified tumor targeted nano drug delivery system is prepared via self-assembly of a slightly acidic environment targeted polypeptide modified dendrimer encapsulated gene, wherein the surface of the dendrimer is rich of amino groups. According to the preparation method, a polypeptide of transmembrane helix protein C derived from bacteria visual purple is used for modifying a high molecular carrier, enrichment and adhesion onto cells is realized via a pH sensitive cell membrane insertion method, and entering into cells is realized via electrostatic adsorption guided endocytosis, so that untaking of tumor cells on drugs is improved, and toxic and side effects are reduced. According to the tumor targeted nano drug delivery system, cell membrane is taken as the target point, and the polypeptide is taken as the target head group in tumor slightly acidic environment, targeting and curing efficiency are high, the preparation method is simple and convenient, and tumor cell drugs, which are derived from human or animal, and is used for targeted therapy, can be prepared.