Abstract: We established a method for simultaneous determination of methylene blue and its metabolites in aquatic products using ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). We extracted the sample with acetonitrile for three times and added hydroxylamine hydrochloride and paratoluenesulfonic acid for efficiency in the meantime, purified it by a PRS SPE column, and eluted by V(Acetonitrile):V(Ammonium acetate, 1molL-1)=1:1. Next, based on Thermo Hypersil Gold C18 column, we used a methanol and 2% formic acid as gradient elution separation to deal with the sample, with the electrospray of mass spectrometry in positive ionization mode and the identifying of methylene blue and its metabolites in selected reaction monitoring (SRM mode). The results show that methylene blue and its metabolites have a good linear relationship within 0.50~100.00 gL-1(R0.99). The separation is finished within 10 min. Average recovery (n=6) of the target compounds at 3 levels (2 gkg-1, 4 gkg-1, 10 gkg-1) is 78.11%~94.40%, while the relative standard deviation (RSD) is 2.12%~8.13%. Besides, the limit of detection (LOD) ranges from 0.40 to 0.60 gkg-1, and the limit of quantity (LOQ) is 2.00 gkg-1. The method is convenient, accurate, easily be applied and popularized, which is suitable for determination and confirmation of methylene blue and its metabolites in aquatic products.