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The harm and impact of the COVID-19 pandemic have highlighted the importance of fast, sensitive, and cost-effective virus detection methods. In this study, we developed a DNA aptamer sensor using nanoparticle-enhanced surface plasmon resonance imaging (SPRi) technology to achieve efficient labeling-free detection of SARS-CoV-2 S protein. We used the same DNA aptamer to modify the surface of the SPRi sensor chip and gold nanoparticles (AuNPs), respectively, for capturing target analytes and amplifying signals, achieving ideal results while greatly reducing costs and simplifying the preparation process. The SPRi sensing method exhibits a good linear relationship (R2 = 0.9926) in the concentration range of 1–20 nM before adding AuNPs to amplify the signal, with a limit of detection (LOD) of 0.32 nM. After amplifying the signal, there is a good linear relationship (R2 = 0.9829) between the concentration range of 25–1000 pM, with a LOD of 5.99 pM. The simulation results also verified the effectiveness of AuNPs in improving SPRi signal response. The SPRi sensor has the advantage of short detection time and can complete the detection within 10 min. In addition, the specificity and repeatability of this method can achieve excellent results. This is the first study to simultaneously capture a viral marker protein and amplify the signal using polyadenylic acid (polyA)–modified DNA aptamers on the SPR platform. This scheme can be used as a fast and inexpensive detection method for diagnosis at the point of care (POC) to combat current and future epidemics caused by the virus. [ABSTRACT FROM AUTHOR]