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The extraction of lutein from marigold petals using a surfactant-based aqueous two-phase system is reported. In this work, the effectiveness of the hydrophilic-lipophilic balance of surfactants on extraction performance for the extraction of lutein from marigold petal powder was demonstrated using aqueous solutions of a wide range of non-ionic surfactants. The response surface methodology was applied to obtain optimised conditions for maximum extraction of lutein. At the optimised conditions (Temperature = 37.5 °C, S/L = 0.00375, and surfactant amount = 1.5% (<i>v</i>/<i>v</i>)), 12.12 ± 0.16 mg/g of lutein was obtained. Furthermore, the surface morphology of marigold petal powder (MPP) was analysed using SEM micrographs. Significant changes in surface morphology were observed which suggested better access of surfactant solution to the targeted biomolecule implanted in the matrix. Finally, the antioxidant activity of the obtained lutein extract was analysed using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Results suggest that the antioxidant activity of the lutein extract obtained by the surfactant-based system is more than that of the lutein extract obtained by organic solvents. The aforementioned results suggest that the lutein can be extracted using a surfactant-based aqueous two-phase system (ATPS)<b>.</b>