A novel approach for green and simple solid-phase extraction of ionizable analytes using cation-exchange resin particles packed in a rotating cotton‐based disk has been developed and utilized for the chemiluminescence determination of ofloxacin in biological fluids (serum and urine). The use of highly-available cation-exchange resin provided effective separation of analyte from complex sample matrixes and its elution by aqueous electrolyte solution without any organic solvents. Ofloxacin ionization in acidic sample solution allowed to provide effective separation of the analyte for subsequent chemiluminescence detection. The cotton‐based disk was characterized by effective wettability. This feature promoted high mass transfer of the analyte to the cation-exchange resin surface at separation step and into elution solution at elution step. The sorption time was decreased to 10 min while the elution time was 5 min. Under the optimal conditions, the detector response for ofloxacin was linear in the concentration range from 9 × 10 ⁻⁸ to 3 × 10 ⁻⁵ mol L ⁻¹. The limit of detection, calculated from a blank test based on 3σ was 3 × 10 ⁻⁸ mol L ⁻¹.