The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well‐dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA‐modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red‐to‐purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8‐17 (a Pb2+‐depedent RNA‐cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well‐dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.