Targeting oxidative stress with inhibiting or boosting the endogenous levels of antioxidants potentially has an outstanding effect in the treatment of oxidative-stress-related diseases. The present work demonstrates the synthesis of quercetin nanoemulsion as one of the potential antioxidants for the treatment of oxidative- stress-related diseases. A quercetin nanoemulsion was prepared using poly(lactic-co-glycolic acid) (PLGA), hyaluronic acid (HA) and emulsifier (Tween-20) through a solvent evaporation technique. The efficiency of the nanoemulsion was evaluated with and without chemical permeation enhancer (CPE). The FT-IR result shows no interaction between quercetin and polymer that proves excellent compatibility. The transdermal delivery ability was evaluated using in vitro release and ex vivo permeation analysis. The transdermal drug-release mechanism was studied by the mathematical model and was found to obey a zero-order, diffusion-controlled mechanism. In vitro toxicity and cell behavior, including cell adhesion, proliferation and cell death of quercetin-nanoemulsion-treated L929 cells, were elucidated by the electrical cell-substrate impedance sensing (ECIS) technique. The produced nanoemulsion showed a high encapsulation efficiency, less toxicity, controlled delivery with enhanced transdermal drug permeation and effective scavenging of free radicals.