Anticancer drugs, such as fluorouracil (5-FU), oxaliplatin, and doxorubicin (Dox) are commonly used to treat colorectal cancer (CRC); however, owing to their low response rate and adverse effects, the development of efficient drug delivery systems (DDSs) is required. The cellular prion protein PrP, which is a cell surface glycoprotein, has been demonstrated to be overexpressed in CRC, however, there has been no research on the development of PrP-targeting DDSs for targeted drug delivery to CRC. In this study, PrP aptamer (Apt)-conjugated gold nanoparticles (AuNPs) were synthesized for targeted delivery of Dox to CRC. Thiol-terminated PrP-Apt was conjugated to AuNPs, followed by hybridization of its complementary DNA for drug loading. Finally, Dox was loaded onto the AuNPs to synthesize PrP-Apt-functionalized doxorubicin-oligomer-AuNPs (PrP-Apt DOA). The PrP-Apt DOA were spherical nanoparticles with an average diameter of 20 nm. Treatment of CRC cells with PrP-Apt DOA induced reactive oxygen species generation by decreasing catalase and superoxide dismutase activities. In addition, treatment with PrP-Apt DOA inhibited mitochondrial functions by decreasing the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, complex 4 activity, and oxygen consumption rates. Compared to free Dox, PrP-Apt DOA decreased proliferation and increased apoptosis of CRC cells to a greater degree. In this study, we demonstrated that PrP-Apt DOA targeting could effectively deliver Dox to CRC cells. PrP-Apt DOA can be used as a treatment for CRC, and have the potential to replace existing anticancer drugs, such as 5-FU, oxaliplatin, and Dox.