The 10-23 DNAzyme is an artificially developed functional oligonucleotide that can cleave RNA in a sequence-specific manner. In this study, we designed a new photo-driven DNAzyme incorporating a photoresponsive DNA overhang complementary to the catalytic core region. The photoresponsive overhang region of the DNAzyme included either azobenzene components (Azos) or 2,6-dimethyl-4-(methylthio)azobenzene units (SDM-Azos) each attached to a d-threoninol linker. When the Azos or SDM-Azos were in the trans form, the photoresponsive DNA overhang hybridized with the DNAzyme, and the RNA cleavage activity was suppressed. cis Isomerization of Azos or SDM-Azos, induced by 365 or 400 nm light, respectively, destabilized the duplex between the photoresponsive overhang and the catalytic core, and the DNAzyme recovered RNA cleavage activity. Reversible photoswitching of the DNAzyme activity was achieved by use of specific light irradiation. Further, light-dependent photoswitching of protein expression in the presence of the DNAzyme was demonstrated. Thus, this photo-driven DNAzyme has potential for application as a photocontrolled gene silencing system and a photoactivatable gene expression system.