To develop a specific, rapid and convenient method based on molecular motor biosensor to detect food-borne rotavirus. A specific probe was encompassed the conservative region of rotavirus’s VP7 segment, and a molecular motor detect device was constructed by connecting probes to F0F1-ATPase molecular motor through biotin-streptavidin system. This biosensor’s sensitivity was 0.005 ng/mL for rotavirus RNA. Extracted virus RNA was conjugated with the biosensor separately, at the same time ATP was synthesized. By comparing fluorescence intensity, we can detect rotavirus RNA in samples. This method possessed specificity for rotavirus, without any cross-reaction with Hepatitis A virus and noroviris, and it could be accomplished within 1 h. We detected 15 samples using this method and the results were compared with RT-PCR results. This method is sensitive and specific for rotavirus, and it can be used to detect food-borne rotavirus.