Cyclobutane pyrimidine dimers (CPDs) are the major DNA photoproducts of thymine-thymine dinucleotides upon ultraviolet (UV) irradiation. Failure in the repair of damaged DNA may lead to DNA replication errors, DNA mutations, and even cell death. Photoreactivation can mediate the repair of UV-induced DNA lesions by photolyases upon UVA (315-400 nm) or blue light (400-500 nm) irradiation. Herein, the UVC (254 nm)-induced DNA damage and photoenzymatic repair of the CPD products were simultaneously monitored by electrochemical impedance spectroscopy (EIS). The UVC-damaged dT20 was first immobilized on the gold electrode, and the specific recognition by the anti-CPD antibody leads to significantly increased EIS signals. The electron transfer resistance (Ret) values were linearly proportional to the concentrations of damaged dT20 ranging from 0.005 to 0.1 μM, and a detection limit of 3.06 nM was achieved. Using surface plasmon resonance, the equilibrium dissociation constant (KD) between the CPDs in dT20 and anti-CPD antibody was estimated to be (3.32 ± 0.31) × 10-12 M, indicating the strong binding affinity. Evidenced by EIS, the CPDs in the damaged dT20 could be repaired by the attached DNA photolyase under UVA (365 nm) photoexcitation, and the detachment of the photolyase from the DNA strand was accomplished after completion of the repair process. The repair efficiency was calculated to be 70.0% by EIS, being consistent with that of 71.4% by UV spectroscopy. The electrochemical method is simple, sensitive and straightforward, holding great potential for assaying other types of DNA lesions and their repair processes.