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A novel, simple and more sensitive spectrophotometric procedure has been developed for the determination of Brilliant blue FCF and Carmoisine in food samples by an aqueous two-phase system (ATPS). After extraction, the absorbance values were measured at 527 and 632 nm for Carmoisine and Brilliant blue FCF, respectively. The effects of different parameters such as, polyethylene glycol (type and amount), pH, salt (type and amount), and temperature were investigated and optimum conditions were established. Linear calibration curves were obtained in the range of 0.1-120 ng mL-1 and 0.2-260 ng mL-1for Carmoisine and Brilliant blue FCF, respectively. Under optimum conditions, detection limit based on three times the standard deviation of the blank (3Sb) was 0.014 ng mL-1 for Carmoisine and 0.017 ng mL-1 for Brilliant blue FCF. The relative standard deviation (RSD) at concentration level of 30 ng mL-1 was 3.17% and 1.87% for Carmoisine and Brilliant blue FCF, respectively. The method was successfully applied to the determination of target analytes in spiked samples with satisfactory results.