In this study, we report the use of a phosphatase to catalyze the formation of nanospheres from a hydrophobic compound (Ada-GFFY-OMe, 2) via a unique mechanism. The nanospheres were stable in aqueous solutions for two weeks. Their size could be controlled by both concentrations of the precursor (Ada-GFFY(p)-OMe, 1) and the enzyme and the nanospheres were characterized by SEM and dynamic light scattering (DLS). We then demonstrated that nanospheres could help the cell-impermeable propidium iodine (PI) to penetrate the cells, which implied that the nanospheres have the potential to be developed into useful carriers for hydrophobic drugs. This study offers the first example of using an enzyme to control the formation of nanospheres from a hydrophobic compound. There is thus the potential that nanospheres can be developed into a carrier for hydrophobic drugs.