Once thought to play a passive role in the storage of genetic information, nucleic acids are now known to be capable of a wide range of functions. Notable examples include allosterically activated ribozyme and deoxyribozyme sensors, RNA aptamers that enhance the fluorescence of small molecule fluorophores, and polymerase ribozymes that can generate transcripts of more than 100 nucleotides in a sequence-directed manner. Discovery of these motifs has been greatly facilitated by a method of artificial evolution called in vitro selection, in which DNA or RNA molecules with useful or interesting functions are isolated from large random sequence libraries using multiple rounds of selection and amplification. Inspired by these examples, in this study we used in vitro selection to identify a novel deoxyribozyme which catalyzes a chemiluminescent reaction. Under optimized conditions this deoxyribozyme generates light more than 10,000-fold more efficiently than the nonenzymatic background reaction. Comparative analysis of more than 100,000 variants obtained using in vitro selection and high-throughput sequencing revealed the secondary structure and sequence requirements of the deoxyribozyme. It also made it possible to design a light-producing sensor in which this deoxyribozyme was used as the signaling component.