Waterborne transmitted viruses pose a public health threat due to their stability in aquatic environments and their ease of transmission with high morbidity rates at low infectious doses. The ability to detect infectious viruses is of critical importance for environmental health and safety. Current methods to assess the presence of infectious viruses are based on detecting the production of cytopathic effects from mammalian cell culture and can take up to weeks before positive identification. Improved methods for rapid and reliable detection and population quantification of infectious viruses are needed for public health assessments. Molecular beacons (MBs), which produce fluorescence upon target binding, provide a simple and separation-free scheme for rapid and sensitive detection of infectious viruses. However, for real-time studies in living cells, the durability of MBs is affected by the intracellular nuclease degradation. Additionally, cell fixation and permeabilization are required to maintain the cellular structure before introducing MBs. In this study, we developed several FRET (fluorescence resonance energy transfer)-based MBs combined with fluorescence microscopy to directly visualize the fluorescent hybrids with newly synthesized viral RNA as an indication of viral infection and to subsequently follow virus spread among cells in situ/in vivo. To prevent nucleolytic degradation, we designed MBs containing 2'-O-methyl RNA bases with phosphorothioate internucleotide linkages, which specifically target the 5' noncoding region of the viral genome. A cell-penetrating Tat peptide was appended to the FRET probes to facilitate non-invasive entry into host cells. To further improve the probe sensitivity in providing real-time and long-term detection of viral replication, MBs composed of quantum dot and gold nanoparticles were generated. Confluent cell monolayers were incubated with the probes followed by infection with virus dilutions and the fluorescence intensity was monitored in real time. Sensitivity experiments showed that 1 PFU detection limit could be achieved within one replicative cycle. The illumination of fluorescent cells increased in a dose-responsive manner and enabled the direct quantification of infectious viral doses. By introducing the modified MBs into the host cell population prior to viral infection and tracking the change of fluorescence signals, we observed cell-to-cell spread when progeny virions infected new host cells in which the infectious cycle could be repeated. The specific nature of these probes enable their utility for rapid diagnostics of viral infections and real-time viral detection in vivo provides sufficient information regarding multiple steps in infection processes at the subcellular level, which will be valuable for the prevention, control, and understanding of viral infection.