The enterovirus 71 (EV71) has threatened Taiwan for more than ten years. Since traditional diagnostic methods are complicated, time consuming, and high-qualified personnel required. Therefore, a new detection process is highly desired. In this study, a high sensitive PC-based electrochemical analyzing system with a functionalized nano-gold modified immunological electrode are developed to detect EV71. Immobilizing specific EV71 polyclone antibodies, which is developed by center for disease control (CDC) Taiwan, onto nano gold of sensing electrode, the affinity interaction of the immobilized antibody with the specific antigen is identified quickly by electrochemical impedance spectrum (EIS) within 20 minutes. The detection limit of this EIS analysis was as low as 50 copy per ml (∼sub-atto molar). In summary, a biosensor and analyzing system based on EIS has been developed to identify EV71 with efficiency, high sensitivity and specificity.