An optimized method was established for determination of 5 isothiazolinone biocides in waterborne coatings. The Optimizer software was used to optimize the parameters such as product ion，fragmentation voltage and collision energy. The influences of the parameters of ammonium acetate and formic acid in eluents，gradients，capillary voltage，dry gas flow rate and nebulizer pressure，acceleration voltage，temperature of dry gas on the mass spectral response was investigated. The samples were diluted with a mixture of water and methanol（1∶1 by volume）or methanol，and homogenized in one-mark volumetric flasks. The extraction was performed in an ultrasonic generator. The extract solution was separated by centrifuge and filtrated. The sample solutions were detected by liquid chromatography-tandem mass spectrometry. The experimental results showed that the linear curve of the 5 isothiazolinones indicated a good linear relationship in the concentration range of more than 98 times dilution. The detection limit of the method was 0. 0013 mg / kg to 0. 42 mg / kg. The recoveries of spiked samples were between 86. 73%and 93. 60%，and the relative standard deviations were 2. 3%~5. 8%.