To rapidly detect quinolones in a water environment, a method combining three-dimensional (3D) fluorescence spectrometry and bilinear least squares/residual bilinearization (BLLS/RBL) is proposed to detect flumequine (FLU), enrofloxacin (ENR), and levofloxacin (LVFX) in water. This method not only accurately analyzes three antibiotics having serious spectral overlap, but also obtains more reliable quantitative prediction results than parallel factor (PARAFAC) method. The average recoveries of FLU, ENR, and LVFX predicted by BLLS/RBL are 98.46%, 99.10%, and 101.69%, respectively. Moreover, the respective root mean square errors are 4.33, 0.33, and 0.26 μg·L ^-1; the sensitivities are 2.8 × 10 ³, 3.5 × 10 ⁴, and 5.5 × 10 ⁴; and the limits of detection are 0.72, 0.06, and 0.03 μg·L ^-1. Results show that the 3D fluorescence spectrum combined with BLLS/RBL provides a reliable method for the detection of quinolones in water.