The invention relates to a preparation method of an iohexol impurity component, namely, 5-(N-(2,3-dihydroxypropyl)acetamido)-N,N'-bis(2,3-dihydroxypropyl)-2,4-diiodo-1,3-benzenedicarboxamide, represented by formula (1). A reference substance is provided for qualitative and quantitative analysis of an iohexol impurity through preparation of the iohexol impurity, so that the quality standard of iohexol is improved, and the important guiding significance is provided for safe medication of the iohexol. The formula (1) is shown in the specification.