DNAzymes as functional units play increasingly important roles for DNA nanotechnology, and fine control of the catalytic activities of DNAzymes is a crucial element in the design and construction of functional and dynamic devices. So far, attempts to control cleavage kinetics can be mainly achieved through varying the concentrations of the specific metal ions. Here we present a facile sequestered DNAzyme beacon strategy based on precisely blocking the catalytic core of the DNAzyme, which can flexibly regulate the DNAzyme cleavage kinetics without changing the concentrations of metal ions. This strategy can be extended to couple with a large number of other RNA-cleaving DNAzymes and was successfully applied in designing a dual stem-loop structure probe for arbitrary sequence biosensing, which provides the possibility of scaling up versatile and dynamic DNA devices that use DNAzymes as functional modules.