The invention discloses a tumor-targeted delivery carrier based on cell-derived micro-vacuoles, a preparation method and an application. The preparation method comprises the following steps of: (A) preparing a conditioned medium: supplementing fetal bovine serum, antibiotics, DSPE-PEG-Biotin and DSPE-PEG-Folate into a basal medium; (B) using the obtained conditioned medium in cell culture, and collecting cell culture supernatant for subsequent separation; (C) carrying out low-speed centrifugation on the obtained culture supernatant to remove cell debris and apoptotic bodies, then adding SA-IONPs, mixing uniformly, incubating, then separating by a magnet, using PBS for re-suspension, eluting for multiple times to obtain the cell-derived micro-vacuoles with membrane surfaces modified by folic acid and iron oxide nano-particles, and freezing for storage; and (D) loading chemotherapeutic drugs or therapeutic genes into the functionalized micro-vacuoles doubly-modified by an electroporation mode, and carrying out re-suspension after separation with the magnet. The tumor-targeted delivery carrier based on cell-derived micro-vacuoles, the preparation method and the application disclosed by the invention are applicable to specific targeting delivery of multiple chemotherapeutic drugs and therapeutic genes, and have the advantages of enhancing the anti-tumor effect, reducing the systemic toxicity and improving the clinical effect of the current therapeutic selection, so that a new hope is brought for clinical therapy of tumors.