<p>Colorectal cancer is one of the most common malignant tumors, and its drug resistance poses a huge challenge and a serious threat to people’s health. In previous studies, we have found that the combination of paclitaxel and BEZ235 has synergistic anti-colon cancer effects, particularly in drug-resistant colorectal cancer, demonstrating excellent anti-tumor effects. Therefore, this study aims to investigate the preparation of the nanoemulsification of paclitaxel and BEZ235 (NEs-PTX-BEZ235) and to explore the potential key parameters for its clinical translation. This study aims to provide a new therapeutic strategy for colorectal cancer, especially for drug-resistant colorectal cancer. The nanomaterials were prepared by low energy self-emulsification method, and analyzed by Malvern laser particle size analyzer. Cell drug uptake was observed under fluorescence microscope. CCK8, Western blot and flow cytometry were used to compare the anti-cancer effects and mechanisms of different experimental groups on ordinary and drug-resistant colon cancer cells. Human colon cancer primary cells were extracted to verify the anti-tumor effect of drugs. Finally, we found that NEs-PTX-BEZ235 significantly killed colon cancer cells, especially drug-resistant cells, and performed better than Nab-PTX. It may play a synergistic role in reducing drug resistance, inhibiting tumor stemness and inducing apoptosis of colon cancer cells by inhibiting drug resistance proteins.</p>