Nervous necrosis virus (NNV), a devastating viral pathogen in grouper aquaculture, severely impacts the grouper farming industry. NNV-induced viral nervous necrosis triggers mass mortality in larvae and juveniles. The 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 is urgent. In this study, we constructed a target-activated aptamer probe (TNA1c-TAA) through structural modification and chemical engineering of TNA1, an aptamer that specifically recognizes NNV-infected cells. 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 nmol/L) precisely detected 1×10³ NNV-infected cells within 1 min at 4–28 . ℃ In vivo detection demonstrated specific recognition of NNV-infected brain tissue cells in grouper, with 100% agreement between the TNA1c-TAA positivity rate and qPCR results. These findings highlight the potential of the TNA1c-TAA for the rapid diagnosis of NNV in aquaculture, laying the foundation for establishing a trinity prevention-diagnosis-control system against aquatic epidemics.