Cells secrete extracellular vesicles (EVs) to mediate precise communication during viral infections, yet the spatiotemporal regulation of EV composition by herpes simplex virus 1 (HSV-1) remains poorly understood. Here, we develop an integrated single-nanoparticle analysis platform combining nanoporous membrane-based EV isolation with an on-chip immunoassay to quantitatively probe EV-HSV-1 interplay throughout infection. A dual-membrane filter design significantly enhances nanoparticle recovery, enabling high-sensitivity single-particle detection. We reveal that HSV-1-infected neural stem cells display viral glycoprotein B on EV surfaces at an early stage (<8 hpi), while intact virions are selectively packaged into EVs later (24-48 hpi). Proteomic profiling indicates infected cell-derived EVs facilitate antigen processing and presentation, potentially amplifying antiviral responses. Functional studies further demonstrate EVs promote viral entry at late stages (48 hpi), likely via EV-virion encapsulation. These findings elucidate a dynamic EV-virus interplay, offering insights into HSV-1 pathogenesis and EV-mediated immune modulation. Our platform provides a transformative approach for advancing infection diagnostics and therapeutics.