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As a new class of green solvents, hydrophobic deep eutectic solvents (DESs) have attracted considerable attention for liquid phase microextraction. In this study, a hydrophobic DES composed of trioctylmethylammonium chloride and oleic acid was successfully applied for the vortex-assisted liquid-liquid microextraction (VA-LLME) of Sudan I in food samples coupled with high performance liquid chromatography with diode array detection (HPLC-DAD). Trioctylmethylammonium chloride and oleic acid at a molar ratio of 1:2 showed higher extraction recovery for Sudan I than other DESs. The deep eutectic solvent was prepared and characterized by Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance spectroscopy (1H NMR). The parameters affecting extraction recovery were optimized in detail. This hydrophobic DES based VA-LLME technique exhibited a high extraction recovery close to 100% at room temperature within 3 min. Under the optimized conditions, wide linear ranges were acquired with r2 equal to 0.9992. The limit of detection was 0.3 µg kg−1. The inter day and intra-day precision values were less than 5.4%. This hydrophobic DES based VA-LLME technique was successfully applied for the determination of Sudan I from duck blood and chili power samples with the satisfactory recoveries from 83.4 to 114.1% and relative standard deviations from 1.5 to 7.6%. In addition, the analytical parameters of this method and other liquid phase microextraction methods were compared. [ABSTRACT FROM AUTHOR]