Luteolin is expected to be an alternative treatment for gout due to its antiinflammatory activity and xanthine oxidase inhibition. However, its poor solubility and permeability through epithelial and gastrointestinal are hampering its formulation and development process. Transfersome is a lipid-based vesicle that is prospective to enhance absorption and penetration of luteolin. Luteolin-loaded transfersome was optimized by response surface methodology (RSM) for four parameters of particle size (Z-average), polydispersity index, zeta potential and entrapment efficiency. Transfersome formula was carried out using central composite design (CCD) and the selected independent variables were total lipid (mixture of phospholipid-Tween 80) and luteolin concentration. Fourteen formulas of luteolin-loaded transfersome were prepared by thin film hydration, followed by sonication method. Total lipid and luteolin concentration significantly affect the entrapment efficiency only. The other parameters were not affected by change of these variables. Optimum formula of 4.88% total lipid and 0.5% luteolin with desirability value of 0.609 has been verified to ensure conformity with prediction parameters. Vesicle image using transmission electron microscope (TEM) revealed spherical particles and occurrence of particle aggregation. Optimum formula of luteolin-loaded transfersome possessed these characteristics: particle size of 286.03±8.46 nm, polydispersity index of 0.480±0.013, zeta potential of -18.67±0.379 mV and entrapment efficiency of 94.97±0.28 %. However, those values did not correspond to the predicted value and was confirmed by the low value of adjusted and predicted R-squared. This method can be applied to optimize the entrapment efficiency and in future, it is applicable in optimizing formula of transfersome by including more variables.