The invention discloses a method for researching migratory behaviour of anguilla japonica by employing otolith microchemistry, wherein the migratory behaviour of anguilla japonica is determined by analyzing the microchemical constitution of the cross section of otolith life history of anguilla japonica by using a proton scanning microprobe technique, and the process comprises otolith excavating, embedding, flaking, film bonding, otolith centering, life history cross section scanning, guide sample correction and life history analysis. The invention provides a method which is capable of accurately positioning the center of otolith, sensitive in detection and quick in analyzing Sr / Ca ratio change in the cross section of the otolith life history, so that the following problems are solved: low Sr element content in otolith, long detection time, big errors, difficulty in accurate positioning because the center of an otolith ground slice of anguilla japonica is not at the geometric center, easiness in loss of information of partial stages of the life history caused by the use of a quick line scanning manner, and the like; and in combination with the annual ring marks of the otolith, the migratory behaviour of anguilla japonica at habitats such as fresh water, river mouths, oceans and the like can be effectively analyzed.