The (6-4) photoproduct, which is one of the major UV-induced DNA lesions formed at bipyrimidine sites, causes carcinogenesis at high frequency. The (6-4) photolyases restore the (6-4) photoproducts to their intact bases in a light-dependent manner, but its overall repair mechanism remains obscure. To investigate the light-dependent conversion of the (6-4) photoproduct, we prepared a (6-4) photoproduct analog with modification at 3' pyrimidone ring, in which the carbonyl group was replaced with an imine to apply to the (6-4) photolyase assay. The (6-4) photolyase shows affinity to an oligonucleotide carrying this imine analog of the (6-4) photoproduct, though the imine analog is not repaired by the (6-4) photolyase.