Methylene blue (MB) is commonly used in aquaculture as a fungicide and antidotes. This study was designed to explore the pharmacokinetics of MB in Japanese eel ( Anguilla Japonica) immersed in 10 mg L −1 and 20 mg L −1 MB for 0.5 h. The concentrations of MB and its derivatives in the blood, liver, kidney, skin and muscle were determined by HPLC after immersion. The results showed that the changes of drug concentrations in Japanese eel were basically the same in different dose groups, showing a general trend of increasing at first and then decreasing, but the peak time was slightly different. The peak concentration of the drug was positively correlated with the dose level. The peak concentrations of MB in MB (20 mg L −1) group, MB, azure A and azure B in the tissues of Japanese eel were significantly higher than those in MB (10 mg L −1) group. Moreover, MB, azure A and azure B remained for a long time and could still be detected at 64 days, and azure C was not detected in tissues.