A sensitive HPLC-FLD method with two-channel fluorescence detection was established and validated for quantification of four fluoroquinolones (norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin) in urine samples. The fluorescent detector was set at two-channel excitation and emission wavelengths: 280/445 nm (for NOR, CIP) and 290/500 nm (for LEVO, MOXI). The measurement protocol was in compatible with both of the sample treatment procedures including the liquid-liquid extraction (LLE) and solid-phase extraction (SPE). The method was well-validated with the limit of quantification: 0.027; 0.034; 0.024; 0.020 µg/mL (for LLE), and 0.028; 0.033; 0.024; 0.020 µg/mL (for SPE) for NOR, CIP, LEVO, MOXI, respectively. The method has been proved to be precise and accurate, with the relative standard deviation lower than 3% and the recovery ranging from 98.0%¸102.0%, for all fluoroquinolones. The proposed HPLC-FLD method provides an alternative approach for the simultaneous analysis of fluoroquinolones in urine.