The high potential of thymoquinone as an ingredient and/or additive in the food, pharmaceutical and cosmetic industries has been well established in previous studies. However, its extraction from natural sources was considered in the limited studies and none of them included the microwave-assisted extraction (MAE) of a thymoquinone-rich extract and process optimization. In the present study, this high value -added bioactive was aimed to extract from its well-known natural source, black cumin seed (Nigel la sativa L.), using methanol as a solvent for all of the studied extraction methods. For extraction of a compound of interest, microwave-assisted extraction system having temperature controlling function was used and its performance was compared with common extraction methods, Soxhlet and conventional solid/liquid extraction. The results indicated that the MAE system provided a rich extract containing thymoquinone, which was 2 and 7 times higher than those produced by conventional solid/liquid extraction and Soxhlet, respectively. Influences of temperature, time and solvent/solid ratio on thymoquinone yield were investigated for MAE. The solvent/solid ratio was found to have the main effect on extraction performance, whereas an interaction effect of temperature and time was significant. Variables of MAE were optimized by response surface methodology to produce a thymoquinone-rich extract. Optimal conditions for the highest yield of thymoquinone were determined as 10 minutes extraction at 30 degrees C, using 30 ml solvent per gram of black cumin seed. The estimated thymoquinone yield of the extract was 628 mg/kg black cumin seed. It could be concluded that the currently optimized MAE with temperature controlling function is a promising technique to produce a thymoquinone-rich extract from black cumin seeds.