Ribonucleic acid (RNA) interference (RNAi) therapies are promising cancer treatment modalities that can specifically target abnormal proto-oncogenes, thus improving the therapeutic effect. For the treatment of pancreatic cancer, targeting one mutant proto-oncogene by RNAi usually does not yield the desired therapeutic efficiency. Both K-ras gene mutations and Notch1 overexpression are common symptoms in pancreatic cancer patients, and play a crucial role in pancreatic cancer cell drug resistance. In this study, biodegradable charged polyester-based vectors (BCPVs) were synthesized for the co-delivery of K-ras and Notch1 small interfering ribonucleic acid (siRNA) into MiaPaCa-2 cells (pancreatic cancer cell line) to overcome drug resistance to gemcitabine (GEM), a first-line chemotherapeutic drug used in the clinic. BCPVs could effectively absorb negative siRNA to form a capsule-like structure, prevent siRNA from nuclease digestion in the serum, and promote effective siRNA cell internalization and endosomal escape. Through K-ras and Notch1 gene silencing in MiaPaCa-2 cells, BCPV-siRNA^K-ras-siRNA^Notch1 nanocomplexes effectively reversed the epithelia-mesenchymal transition (EMT) in MiaPaCa-2 cells, thereby greatly enhancing the sensitivity of MiaPaCa-2 cells to GEM. MiaPaCa-2 cell proliferation, migration, and invasion were effectively inhibited, and cell apoptosis was also significantly enhanced by the synergistic antitumor effect of BCPV-siRNA^K-ras-siRNA^Notch1 nanocomplexes and GEM. These results suggest that this combination RNAi therapy can be used to improve cancer cell sensitivity to chemotherapeutic drugs. Specifically, this newly developed strategy has a great potential for treating pancreatic cancer.