Lipid‐based nanodelivery systems are now widely used in the pharmaceutical and food industries to enhance the delivery of bioactive compounds by leveraging the properties of lipids. Nanostructured lipid carriers (NLC), as one of the lipid‐based nano‐delivery systems, employs a blend of solid and liquid lipids, which improves the solubility, stability, and bioavailability of bioactive substances. This study aimed to optimize the NLC containing thymoquinone‐rich oil (NLC‐TQO) formulation using the Box–Behnken design; assess its antioxidant activity and oxidative stability by measuring 2,2‐diphenyl−1‐picrylhydrazyl (DPPH) radical scavenging activity, thymoquinone (TQ), and tocopherol contents over 28‐day storage; and evaluate the impact of incorporating beta‐carotene as singlet oxygen quencher on the oxidative stability of the NLC‐TQO. The optimized formulation involved 4.16% lipids (glyceryl monostearate and TQO), 5.85% Tween 80 as the surfactant, and 8.20 min of ultrasonication. NLC formulations demonstrated better DPPH activity retention than the emulsions, with cold and dark storage conditions being the most effective for preserving antioxidant activity. TQ stability in the NLC was observed at elevated temperatures without light, with around 50% remaining intact. However, TQ degraded rapidly under light, almost entirely by day 7. Gamma‐tocopherol exhibited better stability than alpha‐tocopherol, especially under light. The addition of beta‐carotene enhanced NLC‐TQO's oxidative stability. These findings revealed the potential of the NLC formulation as an effective delivery system for TQ with better oxidative protection compared to emulsions, making it a promising option for pharmaceutical and food industry applications.