Inactivated Hemagglutinating Virus of Japan Envelope (HVJ-E) was immobilized on electrospun nanofibers of poly(ε-caprolactone) by layer-by-layer (LbL) assembly technique. The precursor LbL film was first constructed with poly-L-lysine and alginic acid via electrostatic interaction. Then the HVJ-E particles were immobilized on the cationic PLL outermost surface. The HVJ-E adsorption was confirmed by surface wettability test, scanning laser microscopy, scanning electron microscopy, and confocal laser microscopy. The immobilized HVJ-E particles were released from the nanofibers under physiological condition. In vitro cytotoxic assay demonstrated that the released HVJ-E from nanofibers induced cancer cell deaths. This surface immobilization technique is possible to perform on anti-cancer drug incorporated nanofibers that enables the fibers to show chemotherapy and immunotherapy simultaneously for an effective eradication of tumor cells in vivo.