5-Hydroxymethylcytosine (5 hmC) is a novel epigenetic modification that plays an important role in mammalian nuclear reprogramming, regulation of gene activity, and initiation of DNA demethylation. In this paper, an electrochemiluminescence sensor was constructed for 5 hmC detection based on thiol functional FeO magnetic beads and covalent chemical reaction of -CHOH in 5 hmC. First, FeO magnetic beads were prepared and modified with thiol. Then, 5 hmC was captured on the surface of the magnetic beads by the reaction between -CHOH of 5 hmC and -SH of the thiol-functionalized FeO under the catalysis of DNA methyltransferase (M. HhaI). After that, through a series of reactions, phos-tag-biotin, avidin, and bis(hexafluorophosphate) (Ru (bpy) (phen-5-NH) (PF)) (Ru) were further successively immobilized on the surface of the magnetic beads. More importantly, these reactions were carried out in a solution to ensure the activity of the biomolecules, and further to ensure that the reaction proceeded sufficiently. Finally, an ECL signal was generated by the introduction of Ru. The concentration of 5 hmC presented a good linear relationship with the ECL signal intensity in the range of 0.01-500 nM, and the detection limit was 2.86 pM. Moreover, we also used this method to study the 5 hmC content change in rice seedlings treated with antibiotics and heavy metal composite pollutants, and in chicken embryo fibroblast cell infected with and without avian leukosis virus subgroup J.