A sensitive and disposable electrochemical impedance biosensor to detect Japanese encephalitis virus (JEV) was developed based on a gold nanoparticle (AuNP)-modified screen-printed carbon electrode (SPCE). A biosensor was fabricated through covalent grafting of a mixed self-assembled monolayer on AuNPs with a specific antibody. To detect JEV and achieve signal amplification, the horseradish peroxidase (HRP)-labeled second antibody was linked to the biosensor through a sandwich immunity reaction. HRP was used to catalyze 4-chloro-1-naphthol oxidation to produce an insoluble precipitate, which introduced a barrier to electron transfer on the electrode. Electrochemical impedance spectroscopy (EIS) was used to monitor the precipitation on the electrode. The electron-transfer resistance (Ret) of the biosensor was directly correlated with the concentration of JEV in the solution. Under optimal conditions, the method generated a linear response range between 500 and 5 × 105 pfu mL-1, and the detection limit was 167 pfu mL-1. The biosensor exhibited good selectivity against other viruses.