The invention provides an active targeting type amphipathic polypeptide nano-drug carrier and preparation and application thereof, and belongs to the technical field of biological medicines. According to an amphipathic polypeptide, an alkyl chain serves as the hydrophobic end, a polypeptide chain with active targeting functional and side chain modification fluorescent functional molecules serves as the hydrophilic end, and an anti-tumor drug is wrapped in a hydrophobic cavity of a micelle formed by self-assembling the amphipathic polypeptide. The nano-carrier can actively target tumor cells and enter the tumor cells through receptor-mediated endocytosis, the amphipathic polypeptide and phospholipid molecules have strong interaction, and the phagocytosis of a nano-drug by tumor cells is promoted. In the process, tracing can be conducted through fluorescent imaging of fluorescent functional molecules modified on the polypeptide chain, and the tumor imaging aim is realized. Finally, the anti-tumor drug is slowly released to kill tumor cells and restrain the growth of tumors. The amphipathic polypeptide nano-carrier is free of toxin, high in biocompatibility and remarkable in anti-tumor efficiency, and the tumor diagnosis and treatment can be integrated.