For the first time, we synthesized the co-delivery nanopolymers using zein protein as the core and chitosan polysaccharide as the shell to deliver curcumin (Cur) and berberine (Ber) in MDA-MB-231 breast cancer cells. It has been shown that Cur and Ber altogether have synergistic effects on multiple cancers. Herein, the curcumin-zein-berberine-chitosan (Cur-Z-Ber-Ch) nanoparticles were fabricated and their organization procedure was reported. Physicochemical properties of synthesized nanoparticles were determined by Fourier transform infrared (FTIR) spectroscopy, XRD and fluorescence spectroscopy analyses. The nanoparticles included relatively small particles (d = 168.24 nm) with +36.76 mV zeta potential. The resulting nanoparticles had high entrapment efficiency (about 75%) for Cur and 60% for Ber. The Cur-Z-Ber-Ch nanoparticles represented ideal redispersibility and storage stability after 4 months. Drug release of the freeze-dried nanoparticles had pH-sensitive characteristic. In vitro cytoxicity assay demonstrated that Cur-Z-Ber-Ch nanoparticles induced elevated cytotoxic effect in MDA-MB-231 and A549 cancer cells. In vitro studies in MDA-MB-231 cells demonstrated that the Cur-Z-Ber-Ch nanoparticles could successfully increase cellular uptake and apoptosis with significant inhibition of IL-8 pro-inflammatory cytokines in comparison to the free Cur + Ber bioactive molecules. These bio-nanoparticles are the co-delivery vehicle for Cur and Ber which could be beneficial for participating them into pharmaceutical products.