An abasic site called dSpacer has been introduced into duplex regions of the 8-17 DNAzyme and adenosine aptamer for label-free fluorescent detection of Pb2+ and adenosine, respectively. The dSpacer can bind an extrinsic fluorescent compound, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), and quench its fluorescence. Addition of Pb2+ enables the DNAzyme to cleave its substrate and release ATMND from DNA duplex, recovering the fluorescence of ATMND. Similarly, the presence of adenosine induces structural switching of the aptamer, resulting in the release of ATMND from the DNA duplex and a subsequent fluorescence enhancement. Under optimized conditions, this label-free method exhibits detection limits of 4 nM for Pb2+ and 3.4 mu M for adenosine, which are even lower than those of the corresponding labeled-DNAzyme and aptamer sensors. These low detection limits have been obtained without compromising any of the selectivity of the sensors. Finally, the dynamic range of the adenosine sensor has been tuned by varying the number of hybridized base-pairs in the aptamer duplex. The method demonstrated here can be applied for label-free detection and quantification of a broad range of analytes using other DNAzymes and aptamers.