Abstract: A method for the rapid determination of dyes for malachite green, leucomalachite green, azure A, azure B, azure C, methylene blue, which are illegally mixed with indigo naturalis is set up. The samples were extracted with 5% formic acid in acetonitrile, and the qualitative and quantitative detections were carried out using ultra high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS) in the multi reaction monitoring mode(MRM). Among them, malachite green and leucomalachite green were quantified using the internal standard method, and azure A, azure B, azure C and methylene blue were quantified using the external standard method. The linear range of the 6 dyes was 1~30 ng/mL, the detection limits of malachite green, leucomalachite green, azure A, azure B, azure C, methylene blue were 0.5、0.5、1.0、1.0、1.0、1.0 ng/g respectively, and the quantitative limits for malachite green, leucomalachite green, azure A, azure B, azure C, methylene blue were 1.0、1.0、2.0、2.0、2.0、2.0 ng/g respectively, the recoveries of the three levels ranged from 79.7% to 119.7%, and the RSD% of precision ranged from 0.7% to 1.8%. The pretreatment of this method is simple, and its sensitivity is high. The method is suitable for the fast detection and analysis of dyes malachite green, leucomalachite green, azure A, azure B, azure C, methylene blue, which are illegally mixed in indigo naturalis.