Gastric cancer (GC) is known as a deadly malignancy all over the world, yet none of the current therapeutic regimens have achieved efficacy. this current study has aimed to optimize and reduce treatment doses and overcome multidrug resistance in GC by developing optimum niosomal formulation for the delivery of doxorubicin (DXR), paclitaxel (PTX), and their co-delivery. The particles' size, polydispersity index (PDI), and entrapment efficacy (EE%) were optimized using statistical techniques, i.e., Box-Behnken and Central Composite Design. In contrast to soluble drug formulations, the release rate of medicines from nanoparticles were higher in physiological and acidic pH. Niosomes were more stable at 4 °C, compared to 25 °C. The MTT assay revealed that the IC₅₀ of drug-loaded niosomes was the lowest among all developed formulations. The apoptosis-related genes (CASPASE-3, CASPASE-8, and CASPASE-9) and tumor suppressor genes (BAX, BCL2) were evaluated in cancer cells before and after treatment. In comparison to control cells and cells treated with soluble forms of DXR and PTX, while the expression of BCL2 decreased, the expression of BAX, CASPASE-3, CASPASE-8, and CASPASE-9 was enhanced in cells treated with drug-loaded niosomes. Drug-loaded niosomes inhibited colony formation capacity and increased apoptosis in human AGS gastric cancer cells. Our results indicate that co-delivery of DXR and PTX-loaded niosomes may be an effective and innovative therapeutic approach to gastric cancer.