Vascular endothelial growth factor (VEGF) is a potential target for cancer treatment because of its role in angiogenesis and its overexpression in most human cancers. Currently, anti-VEGF antibodies have been shown to be promising tools for therapeutic applications. However, large size, poor tumor penetration, immunogenicity, and production in cost- and labor-intensive conditions are major drawbacks of such agents. The antigen-binding regions of camelid single-chain antibodies (VHHs), due to their unique biophysical characteristics, offer an alternative to conventional antibodies for tumor-targeting purposes. The present study was undertaken to generate and characterize anti-VEGF VHHs from an immune VHH library using phage display. Four rounds of panning were performed, and selected VHHs were characterized using various immunological techniques. Assessment of the antigenic profile of VHHs was done using competition enzyme-linked immunosorbent assay (ELISA). Selected VHHs reacted strongly to VEGF in indirect ELISA and cross-reactivity ELISA tests. The binding affinity of three VHHs, ZFR-1, ZFR-2, and ZFR-5, ranged from 2.5 to 80 nM, and among them, ZFR-5, which was selected for proliferation assay, significantly inhibited the endothelial cell growth in a dose-dependent manner. Taken together, our results indicate that ZFR-5 and other VHHs may be promising tools in cancer research and treatment.