Chitosan (CS) nanoparticles of thymoquinone (TQ) were prepared by the ionic gelation method and are characterized on the basis of surface morphology, in vitro or ex vivo release, dynamic light scattering, and X-ray diffractometry (XRD) studies. Dynamic laser light scattering and transmission electron microscopy confirmed the particle diameter was between 150 to 200 nm. The results showed that the particle size of the formulation was significantly affected by the drug:CS ratio, whereas it was least significantly affected by the tripolyphosphate:CS ratio. The entrapment efficiency and loading capacity of TQ was found to be 63.3% ± 3.5% and 31.23% ± 3.14%, respectively. The drug-entrapment efficiency and drug-loading capacity of the nanoparticles appears to be inversely proportional to the drug:CS ratio. An XRD study proves that TQ dispersed in the nanoparticles changes its form from crystalline to amorphous. This was further confirmed by differential scanning calorimetry thermography. The flat thermogram of the nanoparticle data indicated that TQ formed a molecular dispersion within the nanoparticles. Optimized nanoparticles were evaluated further with the help of scintigraphy imaging, which ascertains the uptake of drug into the brain. Based on maximum concentration, time-to-maximum concentration, area-under-curve over 24 hours, and elimination rate constant, intranasal TQ-loaded nanoparticles (TQ-NP1) proved more effective in brain targeting compared to intravenous and intranasal TQ solution. The high drug-targeting potential and efficiency demonstrates the significant role of the mucoadhesive properties of TQ-NP1.