Background: The selection of an eco-friendly, selective and cheap extraction solvent for effective and green liquid-phase microextraction is always a challenging task in analytical chemistry. Supramolecular solvents serving as safer alternatives to traditional extractants hold promise for this goal. Additionally, they can be tailored to a specific task. However, traditional surfactants can still pose environmental risks due to their persistence and toxicity. To address the issue, introduction of bio-based surfactants derived from natural sources to obtain supramolecular solvents is of high interest. Results: Novel supramolecular solvent based on a biodegradable amino acid-based anionic surfactant (sodium cocoyl glutamate) was proposed for liquid-phase microextraction for separation and used for preconcentration of penicillins (ampicillin and amoxicillin) from bovine milk samples. In the developed approach only two polar organic acids and the surfactant were added to a sample phase. Polar organic acids played a dual role: firstly, acetic and trichloroacetic acids were employed for protein precipitation in milk matrix; secondly, the obtained acidic supernatant induced coacervation in a micellar solution of the amphiphile. The analytical performance of the microextraction technique was demonstrated by liquid chromatography-mass spectrometry determination of the analytes in bovine milk with different fat content. The limits of detection were equal to 0.3 and 1 μg kg−1 for ampicillin and amoxicillin, respectively. Significance: In this study, formation of sustainable green supramolecular solvent in the presence of sodium cocoyl glutamate as an amphiphile was shown for the first time. It was established that certain organic acids induced coacervation in a micellar solution of the amphiphile through partial neutralization of the negative surface charge on the supramolecular aggregates.