The first report of on-line coupled supercritical fluid extraction (SFE) with reversed-phase liquid chromatography for the quantitative analysis of analytes in aqueous matrices is described. Two commercial systems (e.g. SFE and HPLC) were connected via a single six-port injection valve. By using water to eliminate residual decompressed CO2 gas in the solid-phase extraction trap, quantitative extraction and transfer were achieved for the target analytes (progesterone, phenanthrene, and pyrene) spiked in water, as well as in real samples (urine and environmental water). During each extraction, no restrictor plugging was realized. Extraction temperature and pressure were optimized. Different amounts of salt were added to the aqueous matrix to enhance ionic strength and thus extraction efficiency. Methanol and 2-propanol were used as CO2 modifiers. Compared with dynamically mixing modifier with the CO2 extraction fluid, pre-spiking the same amount of modifier in the extraction vessel enhanced the recovery approximately 30% for progesterone, phenanthrene, and pyrene due to a "co-extraction effect".