摘要： 5-Hydroxymethylcytosine (5hmC) plays an important role in switching genes on and off in mammals, and it is implicated in both embryonic development and cancer progression. Herein, a novel photoelectrochemical (PEC) biosensor was developed for 5hmC detection based on WS2 nanosheets as the photoactive material and boronic acid functionalized carbon dots (B-CDs) for signal amplification unit. This biosensor can also be used for T4-β-glucosyltransferase (β-GT) activity assessment. Firstly, WS2 nanosheets and gold nanoparticles (AuNPs) were immobilized on an ITO electrode surface. Then probe DNA was immobilized on this electrode surface via Au-S bond. Afterwards, the complementary DNA containing 5hmC was then captured on the modified electrode surface by hybridization. Subsequently, β-GT transferred glucose from uridine diphosphoglucose to the hydroxyl groups of the 5hmC residues. After glycosylation, B-CDs could further be immobilized on the modified electrode surface resulting in a strong photocurrent. The PEC biosensor afforded high selectivity, excellent sensitivity and good reproducibility, with detection limits of 0.0034 nM and 0.028 unit/mL for 5hmC and β-GT, respectively. Results demonstrate that the photoelectrochemical strategy introduced here based on WS2 nanosheets and B-CDs offers a versatile platform for hydroxymethylated DNA detection, β-GT activity assessment and β-GT inhibitor screening.