In this work, a liquid-liquid microextraction approach based on in situ decomposition of deep eutectic solvent and following formation of supramolecular solvent was presented for the first time. The quasi-hydrophobic deep eutectic solvent based on choline chloride and medium-chain fatty acid was mixed with an aqueous phase containing tetrahydrofuran, resulting in the solvent decomposition and medium-chain fatty acid dispersion in the aqueous phase. In the obtained mixture, tetrahydrofuran induced the formation of a supramolecular solvent acting as an extraction solvent. Choline chloride provided a salting-out effect to enhance the mass transfer of the target analyte. The capabilities of the approach were demonstrated in the determination of biotin in dry cat food and brewer's yeast by high-performance liquid chromatography with diode array detection. The proposed microextraction procedure provided high extraction recovery of highly polar analyte – biotin (92 %). The limit of detection, calculated from the blank tests based on 3σ, was 0.3 mg kg−1. It is the first LLME procedure for the determination of biotin.