As a valid tumor marker, vascular endothelial growth factor 165 (VEGF165) is an effective therapeutic target for anticancer treatments. Aptamers hold great promise for the development of anti-VEGF strategies. In this study, anti-VEGF165 ssDNA aptamers were screened using a semirational design and a multilevel screening strategy. Recombinant human VEGF165 protein was used as a target for the construction of an ssDNA virtual aptamer library with ssDNA that had one sole secondary structure. After silicon-assisted prescreening, circular dichroism and isothermal titration calorimetry were used to further screen for candidates. Three aptamers (nos. 524, 529, and 64) with one sole secondary and tertiary structure, showing a high affinity for VEGF165, were identified. The KD values obtained using surface plasmon resonance analysis were 36.3, 288, and 79.3 nM for aptamers 524, 529, and 64, respectively. Cytological tests revealed that the three aptamers inhibit rhVEGF165-induced proliferation of HUVECs. Specifically, aptamer 529 had the strongest inhibitory effect (nearly 100% inhibition). The screening strategy used in our study showed improved screening efficiency relative to other methods and resulted in aptamers with one sole conformation. The aptamers had an advantage in ensuring the uniqueness of aptamer targeting. This semirational design and multilevel screening strategy provide a reference for the screening of other aptamers.