We demonstrate for the first time the development of a universal DNAzyme-based bioluminescent sensor for label-free detection of various biomolecules including DNAzyme and DNA. The presence of DNAzyme may induce the cyclic cleavage of riboadenosine (rA)-containing substrates, and the subsequent digestion of the cleaved substrates by exonuclease III (Exo III) releases abundant AMPs to initiate cyclic AMP pyrophosphorylation-ATP depyrophosphorylation for the generation of an enhanced bioluminescence signal. This sensor can real-time monitor the DNAzyme activity with a detection limit of 3.16 × 10−12 M. Moreover, the DNAzyme may be divided into two subunits for sensitive detection of target DNA. In the presence of target DNA, the two separated subunits may assemble into an active DNAzyme which can catalyze the cyclic cleavage of substrates and initiate the digestion of cleaved substrates by Exo III for the generation of an enhanced bioluminescence signal. This sensor can sensitively detect target DNA with a detection limit of 3.31 × 10−12 M. Importantly, this bioluminescent sensor can achieve a zero-background signal, and its output signal originates from the release of AMP for the generation of self-illuminating light emission without the requirement of either the external labels or the reporting reagents.