Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (Delta G) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, K-d measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. In addition, competition analysis suggested the aptamers shared some common CP binding sites with the anti-CP antibody. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. Compared with controls, mortality of GNNV-infected fish decreased by 40% and 80% after 10 days infection, when the GNNV was pre-incubated with the 1000 nM A10 and B11, respectively. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections. (C) 2016 Elsevier B.V. All rights reserved.