A novel separation/analysis method was developed for the determination of dutasteride in a pharmaceutical formulation and biological samples, which based on surfactant dodecyl sodium sulfate (SDS)/ionic liquid 1-hexyl-3-methylimidazolium bromide (IL, [C(6)min]Br) aqueous two-phase system (ATPS) extraction coupled with spectrofluorimetry. The ATPS was formed owing to new ordered molecular assembly SOS/IL, which not only extracted dutasteride but also sensitized its fluorescence intensity due to synergistic sensitizing effect. Under optimum conditions, a linear calibration curve was obtained in the range of 0.016-0.64 mu g mL(-1) with r = 0.9991 (n = 8). The detection limit was 2.2 ng mL(-1). The preconcentration factor was 7.5. The proposed green analytical procedure was satisfactorily applied to the analysis of dutasteride in dutasteride soft gelatin capsule and human serum with the recovery of 96.7-108.0%. The obtained results of this work were in good agreement with the results of HPLC. The extraction mechanism ATPS-based was also discussed. (C) 2014 Elsevier B.V. All rights reserved.