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Around the world, food-borne virus is one of the main pathogenic microorganisms in the aspect of causing people and animals with acute diarrhoea and poses a serious threat to human health. Therefore, the rapid detection of foodborne virus is very important to guarantee food safety and human health. Here, we reported that we developed a specific, convenient and fast detected method to detect rotavirus (RV) and hepatitis A virus (HAV) by using F0F1-ATPase molecular motor biosensor. Specific RNA probes were encompassed the conservative region of food-borne virus, and a molecular motor detect device was constructed by connecting probes to F0F1-ATPase molecular motor through biotin-streptavidin system. Extracted virus RNA was conjugated with the biosensor separately and meanwhile ATP was synthesized. By comparing their fluorescence intensity, virus RNA level was detected. Our results demonstrated that this biosensor’s sensitivity was the concentrations of 0.005 ng/mL and 0.01 ng/mL for RV and HAV respectively. Furthermore, this method possessed specificity for RV and HAV and none cross-reaction between them. What’s more, this method could be accomplished within 1h. We detected 15 samples by using this method and the results were consistent with RT-PCR results. Overall, this new-typed method based on F0F1-ATPase molecular motor biosensor for RV and HAV detection is sensitive and specific and can be used in the rapid detection of food-borne virus.