© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim The regulation of DNAzyme activity is an important problem for its in vivo applications. We achieved photochemical regulation of DNAzyme activity by using reversible DNA photo-crosslinking of 3-cyanovinylcarbazole (CNVK). The ODN containing CNVK photo-crosslinked to a pyrimidine base in the complementary strand after a few seconds of photoirradiation, and its photoadduct was split by photoirradiation of another wavelength. The activity of photo-crosslinked DNAzyme with CNVK was completely inhibited (OFF state). In contrast, after 312 nm irradiation, DNAzyme activity was recovered upon addition of a substrate strand (ON state). In addition, the photo-crosslinked DNAzyme is prone to enzymatic digestion by exonuclease. This photochemical OFF to ON switching with reversible DNA photo-crosslinking was regulated at the desired time and position; therefore, it might be possible to use it for in vivo application.