A method for the determination and confirmation of methylene blue (MB) in aquatic products was developed. Residues of MB were extracted from homogenized tissues with acetonitrile/sodium acetate buffer solution, and simply cleaned up with dichloromethane liquid/liquid extraction. After concentration and dissolution, the sample solutions were cleaned up by the neutral alumina and weak cation-exchange solid phase extraction (SPE) cartridge, prior to LC-MS/MS analysis. MB was determined at 1.0-20 microg/kg in eel, toasted eel and shrimp, with a limit of quantification of 0.5 microg/kg. Recovery for MB was between 73.0% and 108.3%. This method is fast, exact and sensitive. It can be applied to determine MB in aquatic products.