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Ethosomes are most commonly used vesicular delivery system compared to other lipidic vesicles due to its ethanolic content. Thymoquinone (TQ), the main biologically active complex of Black Cumin seed, has established anticancer activities in several tumors. In this work, the preparation of ethosome for TQ encapsulation by response surface method (RSM) was developed. Central composite design (CCD) was used to optimize three effective parameters involved in ethosome structure including phospholipid, cholesterol and ethanol concentration and the combined effects of them, as well. The obtained optimum values for the variables were phospholipid 5% (W/W), ethanol 45% (V/V) and cholesterol 1.5% (W/W) which were validated by experimental assay. The ethosomal formulation was more characterized for vesicle shape, size, zeta potential and entrapment efficiency percentage. The result showed an efficiency of 99% for drug entrapment with average vesicle size and zeta potential of 20 ± 1 nm and − 63 ± 2 mv, respectively. A quadratic model with a high adequacy (R 2) for size and zeta potential of 0.9319 and 0.9338, respectively, was resulted from RSM and experimental assay. Thymoquinone (TQ) encapsulated in optimized ethosome. Also, cellular toxicity and release test was done. The toxicity and release curves were obtained and the cytotoxic activity of the ethosomic TQ against MCF-7 cell lines was greater than free TQ. IC50 values of free TQ, ethosomic TQ, were found to be 1.10 µg/ml, 0.95 µg/ml, respectively. The generated model suggests a new approach to prediction and experimental lipidic carriers.