Berberine has shown an outstanding antioxidant activity, however the low bioavailability limits its applications in pharmaceutical platforms. Therefore, in this paper, after fabrication of the berberine-HSA nanoparticles by desolvation method, they were well characterized by TEM, SEM, DLS, and FTIR techniques. Afterwards the interaction of HSA and the berberine was evaluated by molecular docking analysis. Finally, the antioxidant activity of the berberine-HSA nanoparticles against H₂O₂-induced oxidative stress in cultured neurons as a model of AD was evaluated by cellular assays. The results showed that the prepared berberine-HSA nanoparticles have a spherical-shaped morphology with a size of around 100 nm and zeta potential value of -31.84 mV. The solubility value of nanoparticles was calculated to be 40.27%, with a berberine loading of 19.37%, berberine entrapment efficiency of 70.34%, and nanoparticles yield of 88.91%. Also, it was shown that the berberine is not significantly released from HSA nanoparticles within 24 hours. Afterwards, molecular docking investigation revealed that berberine spontaneously interacts with HSA through electrostatic interaction. Finally, cellular assays disclosed that the pretreatment of neuronal cultures with berberine-HSA nanoparticles decreased the H₂O₂-stimulated cytotoxicity and relevant morphological changes and enhanced the CAT activity. In conclusion, it can be indicated that the nanoformulation of the berberine can be used as a promising platform for inhibition of oxidative damage-induced Alzheimer's disease (AD).