The invention discloses a superoxide anion probe, and a preparation method and an application thereof. The structural formula of the probe is shown in the description, and I* in the formula is at least one of <123>I, <124>I, <125>I and <131>I. The superoxide anion probe is a nuclear medicine probe compound labeled with radioactive iodine, adopting a trifluoromethanesulfonate group as a response group and using a nucleophilic principle, and can be used for detecting in-vivo superoxide anions; and the superoxide anions have the nucleophilic property of a lone electron, so the superoxide anions can rapidly react with the trifluoromethanesulfonate group in the probe structure, so the probe has a high specificity, and cannot be interfered by other micro-molecules in the in-vivo detection process.