Along with biocompatibility, chemical stability, and simplicity of structural prediction and modification, deoxyribozyme-based molecular sensors have the potential of an improved detection limit due to their ability to catalytically amplify signal. This study contributes to the understanding of the factors responsible for the limit of detection (LOD) of RNA-cleaving deoxyribozyme sensors. A new sensor that detects specific DNA/RNA sequences was designed from deoxyribozyme OA-II [Chiuman, W.; Li, Y. (2006) J. Mol. Biol. 357, 748-754]. The sensor architecture allows for a unique combination of high selectivity, low LOD and the convenience of fluorescent signal monitoring in homogeneous solution. The LOD of the sensor was found to be approximately 1.6 x 10(-10) M after 3 h of incubation. An equation that allows estimation of the lowest theoretical LOD using characteristics of parent deoxyribozymes and their fluorogenic substrates was derived and experimentally verified. According to the equation, "catalytically perfect" enzymes can serve as scaffolds for the design of sensors with the LOD not lower than approximately 2 x 10(-15) M after 3 h of incubation. A new value termed the detection efficiency (DE) is suggested as a time-independent characteristic of a sensor's sensitivity. The expressions for the theoretical LOD and DE can be used to evaluate nucleic acid and protein enzymes for their application as biosensing platforms.