<p>This work describes a simple, sensitive, and environmentally friendly analytical technique for the determining of paracetamol in human urine samples and tablet formulation. The current study for the extraction and enrichment of paracetamol is based on the use of rhamnolipid biosurfactants in emulsion-based liquid-phase microextraction. The separation mechanism of paracetamol is based on the emulsion formation of the biosurfactant-rich phase. First, a bioemulsion solution (colloidal phase) was formed and then the analyte was isolated onto the non-aqueous phase. The second step consists of backextraction of the analyte into an aqueous acceptor phase. Finally, the aqueous acceptor phase was withdrawn using a microsyringe and injected into a liquid chromatography instrument for quantitative analysis. The ability of rhamnolipid biosurfactants to form a stable colloidal phase with regions of different polarities can lead to extraction analyte using van der Waals interactions. Considering the biodegradability of biosurfactants and the removal of chemical surfactants in the sample preparation process, the present method is environmentally friendly. Several influencer factors on extraction efficiency including the amount of rhamnolipid biosurfactant, methanol volume, pH, extraction time, ionic strength, and centrifugation time were investigated and optimized. Under optimal conditions, the enrichment factor for the paracetamol was 160. Also, good linearity was obtained in the range 21–100 µg L⁻¹, with coefficients of determination (<i>r</i>²) ˃ 0.993.</p>