Objective: This study aimed to establish a method for the simultaneous determination of 16 quinolones in freshwater fish by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and then analyze the effect of three different cooking methods (by boiling, steaming and frying) on the residues of quinolone antibiotics. Methods: Samples were ultrasonic extracted with acetonitrile (with 4% formic acid) and purified with C₁₈ solid phase extraction column. The chromatographic separation was performed on a Waters ACQUITY UPLC BEH C₁₈ (1.7 μm, 2.1 mm×100 mm) column, with 0.1% formic acid water-acetonitrile as mobile phase for gradient elution. Mass spectrometry (ESI+) was monitored by multiple reaction ions and quantified by internal standard method. Results: 16 quinolones showed a clear linear relationship between 2.0 ng/mL and 50 ng/mL, and the correlation coefficient was greater than 0.998 49. The limits of detection quantification were 0.3~4.1 μg/kg and 0.9~11.0 μg/kg, respectively. Average recoveries in matrices at low, medium, and high spiked levels ranged from 79.0% to 104.7%, with a standard deviation of 1.0% to 8.1%. There was no significant difference in the residues of 16 quinolones between the fish meat-positive samples of three different cooking methods and the control. Conclusion: The method was simple, rapid, and sensitive, and met the high-throughput screening and dietary risk assessment of quinolone antibiotics in freshwater fish.