Sparassis crispa and Phellinus linteus are edible/medicinal mushrooms that have remarkably high contents of beta-(1-->3)-D-glucan, which acts as a biological response modifier, but difficulty in cultivating the fruiting bodies and extraction of beta-D-glucan have restricted detailed studies. Therefore, a novel process for nanoparticle extraction of Sparan, the beta-D-glucan from Sparassis crispa, and Phellin, the beta-D-glucan from Phellinus linteus, has been investigated using insoluble tungsten carbide as a model for nanoknife technology. This is the first report showing that the nanoknife method results in high yields of Sparan (70.2%) and Phellin (65.2%) with an average particle size of 150 and 390 nm, respectively. The extracted Sparan with beta-(1-->3) linkages showed a remarkably high water solubility of 90% even after 10 min of incubation at room temperature. Therefore, it is likely that this nanoknife method could be used to produce beta-D-glucan for food, cosmetic, and pharmaceutical industries.