In the absence of treatment, detection of pathogenic RNA viruses and quarantine of infected individuals are critical for controlling the infection spread. Conventional antibody-based methods often lack the required accuracy and sensitivity, due to the need for a substantial amount of viral antigens. Similarly, traditional reverse transcription real-time polymerase chain reaction methods face challenges in providing timely results due to their multiple thermal nucleic acid amplification steps. To overcome these limitations, a new method based on imaging of virus-specific DNA markers is developed. This approach employs specially designed single-stranded circular DNA probes that capture virus-derived RNA fragments generated by RNase digestion of the viral genome. These fragments serve as primers for a subsequent single-step DNA filling reaction, producing double-stranded virus-specific marker molecules. These individual markers are recognized through fluorescence imaging following linearization by enzyme cleavage and subsequent fluorescence staining. This method can detect viruses at a genome equivalent level of 14 within 40 min. In addition, the molecule-level imaging-based method effectively detects human immunodeficiency virus-1 in clinical samples. This diagnostic approach does not require sophisticated thermal controls nor extensive nucleic acid amplifications, allowing for accurate, sensitive, and rapid detection without the need for large equipment, offering substantial potential for point-of-care applications.