Polyamide 6 nanofiber polymers were used as modern sorbents for on-line solid phase extraction (SPE) coupled with liquid chromatography. The on-line SPE system was tested for the determination of bisphenol A in river water samples. Polyamide nanofibers were prepared using needleless electrospinning, inserted into a mini column cartridge (5 x 4.6 mm) and coupled with HPLC. The effect of column packing and the amount of polyamide 6 on extraction efficiency was tested and the packing process was optimized. The proposed method was performed using a 50-mu L sample injection followed by an on-line nanofibrous extraction procedure. The influence of the washing mobile phase on the retention of bisphenol A during the extraction procedure was evaluated. Ascends Express C18 (10 cm x 4.6 mm) core-shell column was used as an analytical column. Fluorescence detection wavelengths (lambda(ex) = 225 nm and lambda(em), = 320 nm) were used for identification and quantification of Bisphenol A in river waters. The linearity was tested in the range from 2 to 500 mu g L-1 (using nine calibration points). The limits of detection and quantification were 0.6 and 2 mu g L-1, respectively. The developed method was successfully used for the determination of bisphenol A in various samples of river waters in the Czech Republic (The Ohre, Labe, Nisa, Upa, and Opava Rivers).