The limitations of synthetic drugs, which are commonly characterized by limited bioavailability and poor water solubility, make treating dermatological conditions difficult. According to recent research, β-sitosterol (β-ST), a naturally occurring substance known for its strong anti-inflammatory and antioxidant qualities, may be used as a treatment for conditions affecting the skin. This study explores the encapsulation of β-ST within cubosomal nano-formulations as a solution to the problems of bioavailability and solubility related to hydrophobic bio actives. Glyceryl monooleate (GMO) and P 407 (P407) were used as the lipid phase and Poly vinyl alcohol (PVA) as the aqueous phase in the emulsification process utilized to create CBs. The physicochemical characteristics of the Cubosomes (CBs) loaded with β-ST were thoroughly examined. Particle size, zeta potential, scanning electron microscopy (SEM), encapsulation efficiency, and differential scanning calorimetry (DSC) evaluations were all part of the characterization process. A uniform distribution was suggested by the experimental data, which showed an average particle size of 139.7 nm, a zeta potential of −15.17 mV, and a low polydispersity index (PDI) of 0.225. SEM imaging verified the improved formulation’s cubic structure and nanoscale dimensions. The formulation also showed a positive yield and great encapsulation efficiency. Ascorbic acid was used as a comparison standard in the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) experiment to evaluate the antioxidant activity of the β-ST-loaded CBs. According to the study’s findings, cubosomal encapsulation offers a viable method to improve antioxidant activity, and facilitating regulated medication release—all of which eventually contribute to long-term storage stability.