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Dip-pen nanolithography (DPN) is a widely used technique to create nanoscopic patterns of many different materials. F0F1 -ATPase is a nanoscale rotary molecular motor, and could be used as a biosensor or an ideal motor device in a micro-/nanosystem. In this paper, the DPN technique was used to create nanoarrays of F0F1 -ATPase within chromatophore on a gold surface. The feature size of our F0F1 -ATPase patterns was an average of 130 nm, and mathematically, there were no more than ten F0F1 -ATPases in each dot. The biological activity of patterned F0F1 -ATPase was demonstrated by its adenosine triphosphate synthesis, which was indicated by the fluorescence change of labeled F1300. The patterned F0F1 -ATPase nanoarrays were further constructed as biosensors to detect H9 influenza A virus. The results showed that the biosensor arrays had a remarkable S/N ratio and excellent specificity. This type of biosensor arrays can be further used in high-throughput, high-sensitive detection in future. Meanwhile, the precise patterning of F0F1 -ATPase with desired size, position, and biological activity would accelerate its application in many fields.