Nervous necrosis virus (NNV), one of the most devastating viral pathogens in grouper aquaculture, causes severe impacts on the grouper farming industry. NNV-induced viral nervous necrosis (VNN) triggers mass mortality in larvae and juveniles. There is an urgent need to develop novel detection technologies with on-site rapid response capability, operational simplicity, and high sensitivity/specificity to meet the demands for early diagnosis and precise prevention?. In this study, we innovatively constructed a target-activated aptamer probe (TNA1c-TAA) through structural modification and chemical engineering of TNA1, an aptamer capable of specifically recognizing NNV-infected cells?. Systematic evaluations using flow cytometry, confocal microscopy, and real-time quantitative PCR (qPCR) revealed that，TNA1c-TAA exhibited high specificity for NNV-infected cells without cross-reactivity to other virus-infected cells?. The TNA1c-TAA probe (500 nM) achieved precise detection of 1×103 NNV-infected cells within 1 minute across 4-28℃?. In vivo detection demonstrated specific recognition of NNV-infected brain tissue cells in grouper, with 100% concordance between TNA1c-TAA positivity rate and qPCR results?. The findings validate TNA1c-TAA's potential for rapid diagnosis of NNV disease in aquaculture, laying the foundation for establishing a trinity prevention-diagnosis-control system against aquatic epidemics?.