As a programmable biopolymer, DNA has shown great potential in the fabrication and construction of nanometer-scale assemblies and devices. In this report, we described a strategy for efficient manipulation of gold nanoparticle-bound DNA using restriction endonuclease. The digestion efficiency of this restriction enzyme was studied by varying the surface coverage of stabilizer, the size of nanoparticles, as well as the distance between the nanoparticle surface and the enzyme-cutting site of particle-bound DNA. We found that the surface coverage of stabilizer is crucial for achieving high digestion efficiency. In addition, this stabilizer surface coverage can be tailored by varying the ion strength of the system. Based on the results of polyacrylamide gel electrophoresis and fluorescent study, a high digestion efficiency of 90+% for particle-bound DNA was achieved for the first time. This restriction enzyme manipulation can be considered as an additional level of control of the particle-bound DNA and is expected to be applied to manipulate more complicated nanostructures assembled by DNA.