Controlling chemical and biochemical reactions on nanoscale at the single-molecule level is of great interest due to its possibility for exploring new phenomena and/or unraveling novel mechanisms that are inaccessible for traditional bulk systems. In this paper, we report how DNase I, a well-known and widely used nonspecific endonuclease, digested DNA at controlled sites by using an approach termed combined-dynamic dip-pen nanolithography (CDDPN). High efficient digestion has been realized even under an ambient condition on the surfaces. We attribute this phenomenon to a high effective concentration of DNase I when confined in the nanospots.