Quercetin is a flavonoid with antioxidant and neuroprotective potential for Alzheimer’s disease (AD); however, its clinical applicability is limited by extremely low aqueous solubility and oral bioavailability. To overcome these limitations, a nano-suspension (QT-NS) was developed via bead nano-milling and optimized using Design of Experiments (DoE). Five cyclodextrin derivatives were prescreened at various molar ratios, and β-cyclodextrin (β-CD) was selected as the optimal solubilizer. D-optimal and I-optimal designs were evaluated to optimize stabilizer concentration and drug-to-solubilizer molar ratio. I-optimal demonstrated superior predictive performance and formulation efficiency. The formulation exhibited a particle size of 201.1 ± 3.70 nm, PDI of 0.208 ± 0.02, solubility of 5.708 ± 0.03 mg/mL, and zeta potential of −17.1 ± 2.09 mV, whereas D-optimal design showed a large particle size and low solubility. PXRD analysis confirmed amorphous state of quercetin in the optimized formulation. Dissolution studies revealed significantly improved and pH-independent behavior compared to pure quercetin. In an in vitro hCMEC/D3 blood-brain barrier (BBB) model, the optimized nano-suspension showed approximately 9–10-fold higher permeability than pure quercetin. Cellular uptake analysis using Nile red confirmed increased intracellular internalization. Furthermore, biodistribution analysis at 24 h post-administration of the nano-suspension revealed enhanced brain accumulation.