Enveloped viruses have caused the majority of epidemics and pandemics over the past decade. Direct sensing of virus particles (virions) holds great potential for the functional analysis of enveloped viruses. Here, we explore a series of viral membrane-targeting amphipathic helical (AH) peptide-based molecular probes for the assessment of infectious titers of the human coronavirus 229E virus (HCoV-229E). The M2-protein-derived AH peptide is identified as a strong binder for HCoV-229E, and its conjugate with Nile Red, M2-NR, exhibits fluorogenic response upon selective binding to the viral membrane of HCoV-229E. We demonstrate that the response of M2-NR toward the HCoV-229E virus enables the rapid, simple, and reliable assessment of the infectivity of HCoV-229E. In addition, the present fluorescence assay for infectivity analysis is applicable to various kinds of enveloped virus including influenza A virus, herpes simplex virus-1, and lentivirus.