Accurate determination of the concentration and viability of the viral vaccine vectors is urgently needed for preventing the spread of the viral infections, but also supporting the development and assessment of recombinant virus-vectored vaccines. Herein, we describe a nanoplasmonic biosensor with nanoscale robot hand structure (Nano RHB) for the rapid, direct, and specific capture and quantification of adenovirus particles. The nanorobot allows simple operation in practical applications, such as real-time monitoring of vaccine quantity and quality, and evaluation of vaccine viability. Modification of the Nano RHB with branched gold nanostructures allow rapid and efficient assessment of human adenovirus viability, with ultrahigh detection sensitivity of only 100 copies/mL through one-step sandwich method. Nano RHB detection results were consistent with those from the gold standard median tissue culture infectious dose and real-time polymerase chain reaction assays. Additionally, the Nano RHB platform showed high detection specificity for different types of viral vectors and pseudoviruses. Altogether, these results demonstrate that the Nano RHB platform is a promising tool for efficient and ultrasensitive assessment of vaccines and gene delivery vectors.