Objective To establish a method based on molecularly imprinted solid-phase extraction and liquid chromatography-mass spectrometry(MISPE-HPLC/MS2) for the determination of malachite green,crystal violet,brilliant green and their metabolites in aquatic.Methods The aquatic samples were extracted with acetonitrile and the extract were cleaned up by Alumina-N and molecularly imprinted solid-phase extraction catridge(MISPE).The samples were separated on a Waters Atlantis T3 column(2.1 mm×150 mm,3 μm),acetonitrile and 0.1% formic acid-water were used as mobile phases for isocratic elution,ESI positive ion scan mode was used with selected reaction monitoring(SRM,MS2),and MG-D5 and CV-D6 were used as internal standards for quantification.Results The leaner relation for the malachite green,crystal violet and brilliant green were in the range of 0.2-10 μg/L with a correlation coefficient not less than 0.99.The mean recoveries of spiked samples at the level of 1.0,2.0 and 4.0 μg/kg were in the range of 93.2%-105.9%,92.7%-107.5% and 60.6%-94.4% for malachite green,crystal violet and brilliant green,respectively,the relative standard deviations all less than 12.5%.The detection limits of malachite green,crystal violet and brilliant green were 0.02,0.03 and 0.03 μg/kg,and the quantification limits were 0.07,0.10 and 0.10 μg/kg,respectively.Conclusion This method used the stable isotope dilution technique and matrix-matched calibration was validated to be high extraction selectivity,sensitivity and better purification for the determination of malachite green,crystal violet,brilliant green and their metabolites in a large of real aquatic samples.