Ocular neovascularization is a devastating pathology observed in numerous ocular diseases and is a major cause of blindness. However, all current treatments have their limitations. Hence, it is important to explore new therapeutic strategies. This study aimed to investigate the role of anlotinib, a small molecular multi-targeting tyrosine kinase inhibitor, in ocular neovascularization. Anlotinib administration did not induce any cytotoxicity and tissue toxicity at the tested concentrations. Cellular functional experiments demonstrated that anlotinib inhibited the viability, proliferation, migration, and tube formation ability of endothelial cells (ECs) and pericytes. Western blot analysis demonstrated that anlotinib significantly inhibited the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor β (PDGFR-β), as well as their downstream signaling pathways stimulated by VEGF or PDGF-BB, in a concentration-dependent manner in ECs and pericytes. Using an oxygen-induced retinopathy (OIR) model, our results demonstrated that injection of anlotinib reduced avascular areas and pathological neovascular tufts. Furthermore, using a laser-induced choroidal neovascularization (CNV) model, we observed that the combined treatment of anlotinib and Lucentis reduced the size and thickness of CNV lesions compared to Lucentis monotherapy alone. Taken together, our results suggest that anlotinib could be a promising drug candidate for ocular neovascularization.