A novel method for DNA enzymatic cleavage assays using molecular beacons (MBs) as the substrate for nuclease is described. An MB is a hairpin-shaped DNA probe that is labeled with a fluorescent dye at one end and a quencher at the other end. The loop sequence of the MB can be used as the substrate for single-stranded specific nucleases, whereas the stem of the MB can be designed as the substrate for restriction enzymes. The enzymatic cleavage breaks the MB into fragments and leads to the distance separation of the quencher and the fluorophore, resulting in an increase in the fluorescent signal. Up to an 80-fold signal-to-noise ratio was observed when these probes were cleaved by nucleases. Taking advantage of the MB's detection-without-separation property, this method allows for the real-time detection of DNA cleavage, which is useful for the characterization of DNA nuclease activity as well as the study of steady-state cleavage reaction kinetics. With its simplicity, convenience, high sensitivity, and excellent reproducibility, this method has the potential to be used in the study of both natural and artificial nucleic acid-cleaving enzymes.