A bifunctional sulfonated polyaniline nanofiber mat (NFM) was synthesized by oxidative polymerization and by using polyacrylonitrile nanofiber mat (NFM) as the template. The adsorption capacity of the NFM for fluoroquinolones (FQs) is distinctly improved and the adsorption was a spontaneous process. According to theoretical calculations, hydrogen bonding and ion-exchange interaction are the two major kinds of interaction mechanisms between adsorbent and FQs. The adsorption and desorption properties for FQs were systematically evaluated by adsorption isotherms and by thermodynamic and kinetic studies. The results indicate that the NFM is a viable sorbent for FQs because of rapid mass transfer and good adsorption/desorption efficiency. The NFM is re-usable for at least 20 cycles without decline in performance. Following desorption of the FQs with 10% (V/V) formic acid/acetonitrile, they were quantified by UPLC with MS/MS detection. The sorbent was applied to the solid phase extraction of the FQs norfloxacin, ciprofloxacin, ofloxacin, enrofloxacin, danofloxacin, pefloxacin, marbofloxacin, lomefloxacin and difloxacin from water and biological fluids. Figures of merit include (a) low limits of detection (0.5-1.5 ng L for water, 0.016-0.052 μg L for urine and serum), (b) high recoveries from spiked samples (82.3%-109.4%) with low relative standard deviations (1.1%-12.3%); (c) short extraction times (2 min), and (d) low adsorbent dosage (4 mg). Graphical abstractSchematic representation of a bi-functional sulfonated polyaniline nanofiber mat (NFM) for solid phase extraction (SPE) of fluoroquinolones (FQs) in water, urine and serum.