A photoelectrochemical method was proposed for DNA hydroxymethylation determination using black TiO2 (B-TiO2) nanosphere as photoactive material and ZnO as photoactivity inhibitor. After hydroxymethylated DNA (5hmC-DNA) was captured on the probe modified B-TiO2/ITO electrode surface through hybridization, a glycosyl can be then transferred from uridine diphosphoglucose to 5hmC-DNA and formed a covalent structure with -CH2OH in the presence of T4 β-glucosyltransferase (β-GT). Afterwards, based on a series of covalent reaction, amino functionalized ZnO nanoparticles are further immobilized to the surface of the electrode. Due to the capacity to expend the irradiation light and the photogenerated electron of electron donor, the modified ZnO nanoparticles can result in a decreased photocurrent. The developed method shows wide linear ranges from 0.05-200 nM for hydroxymethylated DNA and 1-220 unit·mL-1 for T4-β-glucosyltransferase. The corresponding determination limits were 0.013 nM and 0.24 unit·mL-1, respectively. The enzyme activity inhibited by 4-phenylimidazole was evaluated. This photoelectrochemical method shows high specificity for 5hmC-DNA (compared to 5fC, 5mC, m6A, control) and β-GT (compared to β-AGT, UGT2B7), and shows excellent stability for testing 5hmC (RSD = 2.75%). Graphical abstractSchematic representation of photoelectrochemical method for DNA hydroxymethylation and β-glucosyltransferase detection based on the glycosylation reaction of -CH2OH in 5-hydroxymethylcytosine and the inhibition activity of ZnO to the photoactivity of black TiO2 nanospheres.