Resistance to apoptosis is a key mechanism underlying how cancer cells evade tumor therapy. Autophagy can prevent anticancer drug-induced apoptosis and promote tumor resistance. The purpose of this study was to improve the sensitivity and efficacy of chemotherapeutic drugs through the inhibition of autophagy. Hydrophobic doxorubicin-hydrazone-caproyl-maleimide (DOX-EMCH) and autophagy-inhibiting si-Beclin1 were simultaneously delivered the amphiphilic peptide micelle system (Co-PMs) using poly(L-arginine)-poly(L-histidine)-DOX-EMCH as the copolymer building unit. The constructed micelle system promoted the escape of si-Beclin1 from endosomes and the release of DOX into the nucleus. The Co-PMs exhibited 2.7-fold higher cytotoxicity and proapoptotic ability in PC3 cells than DOX treatment alone, demonstrating that si-Beclin1 could inhibit the autophagic activity of prostate cancer (PCa) cells by targeting the type III PI3K pathway and enhance the sensitivity of the cells to the chemotherapeutic drug DOX. In addition, the peptide micelles successfully passively targeted DOX and si-Beclin1 to the tumor tissue. Compared with DOX or si-Beclin1 treatment alone, the Co-PMs showed a 3.4-fold greater tumor inhibitory potential , indicative of a significant antiproliferative effect. Our results suggested that the Co-PMs developed in this study have the potential to combine autophagy inhibition and chemotherapy in cancer treatment, especially for PCa.