Stir flat sheet membrane liquid phase microextraction procedure for the chemiluminescence determination of polar antibiotics (ofloxacin and fleroxacin) in human urine samples was developed. The microextraction procedure assumed simultaneous ion-pair complex formation between antibiotic and anionic dye in acidic medium, dispersion of extraction solvent by rotation of a stir flat sheet microporous membrane unit, mass-transfer of complexes from sample into microdroplets of extraction solvent followed by extract collection into the unit. After preconcentration the unit was immersed in alkaline aqueous solution for analytes re-extraction based on ion-pair complex decomposition followed by the chemiluminescence analysis of obtained aqueous phase. The developed microextraction procedure allowed to exclude matrix effects for the selective, simple, and rapid chemiluminescence determination of ofloxacin and fleroxacin in urine samples. The calibration graphs were linear over the concentration ranges of 0.1–25 mg L⁻¹ for ofloxacin and fleroxacin. The LOD values, calculated from the blank tests based on 3σ, were 30 µg L⁻¹ for ofloxacin and fleroxacin. The procedure was conveniently applied to the determination of fluoroquinolones in urine samples.