Abstract: A method for the determination of ofloxacin, pefloxacin, norfloxacin and lomefloxacin in flatfish by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established. Accoding to SN/T 1751.2-2007 Determination of Multi-residues of Quinolones in Food of Animal Origin for Import and Export-Part 2: LC-MS/MS Method, a mathematical model for uncertainty evaluation was established and the sources of uncertainty were analysed. The expanded uncertainty was 1.04, 1.09, 1.34 and 0.99 μg/kg(k=2) for ofloxacin, pefloxacin, norfloxacin and lomefloxacin of contents of 9.26, 10.08, 9.73 and 9.57 μg/kg in flatfish, respectively. As a result, the fitting of the standard curve, the high performance liquid chromatography-tandem mass spectrometry and the preparation of standard solution were the main sources of uncertainty.