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Rhamnolipid (RL) is a surfactant biosynthesized by Pseudomonas aeruginosa strains. The RL feasibility as a high-efficient emulsifier in increasing the physical stability of sesame oil (SO)-based beverage emulsions formed by the ultrasound-assisted emulsification process was studied. The concentration of SO (2.02%–6.98% wt/wt), and RL (0.49%–1.51% wt/wt) was adequately optimized based on the analysis of physical stability, turbidity loss rate (TLR), size index, density, and viscosity characteristics using the central composite rotatable design-response surface methodology. The overall optimum region to attain the highest stability (92.64%), viscosity ratio (0.338), and density (1.017 g/cm³), and the lowest TLR (0.216 Ǻ/day) and size index (0.240) based on the adequate second-order polynomial models (R² = 0.956–0.991) was obtained at the combined level of 2.75% (wt/wt) SO and 0.662% (wt/wt) RL. No significant difference between the observed and predicted values fitted to secondorder polynomial models was found. RL as an efficient biosurfactant can stabilize food-grade emulsion systems. [ABSTRACT FROM AUTHOR]