Light-induced oxidative damage of DNA by O-1(2) generated from photoexcited C-60 was observed at the single-molecule level by atomic force microscopy (AFM) imaging. Two types of DNA origami with uniform morphologies were immobilized on a mica surface and used as DNA substrates. Upon visible light irradiation (528 nm) in the presence of a C-60 aqueous solution, the morphology changes of DNA origami substrates were observed by time-lapse AFM imaging at the single-molecule level by tracking a discrete DNA molecule. The origami showed nicked and flattened morphologies with relaxed features caused by the covalent cleavage of the DNA strands. The involvement of O-1(2) in the on-surface DNA damage was clearly confirmed by AFM experiments in the presence of a O-1(2) quencher and ESR measurements with a spin-trapping agent for O-1(2). This study is the first example of single-molecule observation of oxidative damage of DNA by AFM with corresponding morphology changes in a photocontrolled and time-dependent manner by O-1(2) generated catalytically from photoexcited C-60.