•A procedure to prepare MIP-hollow fibers for fluoroquinolones (FQs) is proposed.•A selective microextraction methodology (MIP-HFM) for FQs has been developed.•Environmental and biological aqueous samples were tested.•Hydrophilic analytes were selectively extracted in non-polar and polar protic media. In the present work, the preparation of a new selective molecularly imprinted polymer (MIP) for the family of fluoroquinolones (FQs) in the pores of polypropylene hollow fibers (HFs) is proposed. The resulting MIP-HFs, which combine solid-phase microextraction (SPME) and molecular imprinting technologies, were used to develop a selective microextraction methodology (MIP-HFM) to determine selected FQs danofloxacin, norfloxacin, enrofloxacin and ciprofloxacin in real samples of environmental and biological interest. Measurements during the optimization of the MIP-HFM and its application to the analyses of real samples were performed by HPLC-UV and HPLC-MS/MS. In order to establish optimum rebinding conditions, the effect of key experimental parameters such as loading media, extraction time and stirring-rate were studied. The developed MIP composites exhibited recognition properties towards the selected hydrophilic antibiotics in non-polar media (toluene) and in polar protic systems such as methanol and methanol/water solutions, up to 20% water content. Recoveries by the developed method for all FQs tested in surface water, groundwater and urine spiked with the analytes of interest at two different concentration levels were within 9.4–24.5 %, with a relative standard deviation, generally <20% (n = 3). The detection limits were within 0.1–10 μg L−1, depending upon the antibiotic and the type of sample.