Pathological retinal neovascularization and choroidal neovascularization are major causes of vision loss in a variety of clinical conditions, such as retinopathy of prematurity, age-related macular degeneration, and diabetic retinopathy. Pigment epithelial-derived factor (PEDF) has been found to be the most potent natural, endogenous inhibitor of neovascularization, but its application is restricted because of its instability and short half-life. Polyethylene glycol (PEG) has been used as a drug carrier to slow clearance rate for decades. The present study investigated PEGylated-PEDF for the first time and evaluated its long-term effects on preventing angiogenesis in vitro and in vivo. PEG showed lower cytotoxicity to human umbilical vein endothelial cells (HUVECs). In vitro, PEGylated-PEDF inhibited HUVEC proliferation, migration, tube formation, and vascular endothelium growth factor secretion and induced HUVEC apoptosis in a dose-dependent manner, and it showed a statistically significant difference compared with the PEDF treatment group. In vivo, PEGylated-PEDF had a long-lasting effect in both plasma and retinal concentrations. In an oxygen-induced retinopathy model, one intravitreous injection of PEGylated-PEDF after mouse pups were moved into room air resulted in a significant difference in the inhibition of retinal neovascularization, which decreased the nonperfusion area, compared with the PEDF-treated group. Our present study demonstrated for the first time the long-term inhibitory effects of PEGylated-PEDF on the prevention of neovascularization in vitro and in vivo. These data suggest that PEGylated-PEDF could offer an innovative therapeutic strategy for preventing retinal neovascularization.