© 2020 Elsevier B.V.In this study, a new deep eutectic solvent (DES) consist of tetrabuthylammonium bromide (TBABr) and 1-octanol at 1:2 M ratio was prepared for the first time and characterized by Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) techniques. The new DES was used as an extraction solvent in the ultrasound assisted liquid-liquid microextraction (UA-LLME) of Erythrosine (E127) in blood, urine, pharmaceutical tablet and syrup samples. Accurate and sensitive determination of erythrosine was accomplished with the combination use of DES-UA-LLME and UV–Vis spectrophotometric detection. Before applying UA-LLME, while protein precipitation was applied to blood samples, pharmaceutical tablets were homogenized and dissolved in methanol. The proposed DES-UA-LLME/UV–VIS procedure was applied directly to urine, syrup sample and supernatant of blood and tablet samples with high recoveries in range of 90% and 100%. Erythrosine in the aqueous sample phase was extracted into 200 μL hydrophobic DES phase at pH 7.0. The effect of important analytical variables such as pH of sample solution, mol ratio of DES components, volume of DES, ultrasonic-based extraction time, sample volume and salt effect were optimized. The preconcentration factor (PF), limit of detection (LOD), intra-day and inter-day relative standard deviations (RSD, %) for the developed procedure were found as 40, 3.75 μg·L−1, 2.6% and 4.6%, respectively.