The manufacture of large-scale and highly ordered fluorescent assemblies has received more and more scientific attention in recent years. An ingenious and low-cost strategy for constructing large-scale DNA nanoarrays by rolling circle amplification (RCA) and a simplified DNA origami technique is proposed in this study. Thrombins are used to trigger the excitation of the fluorescent groups modified on the aptamer staple strands of nanoladders, which leads to the delicate construction of millimeter large-scale fluorescent nanoarrays, whose fluorescence intensity could be effectively regulated by the concentration of thrombin. The above fluorescent nanoarrays will generate a potential application value in the fields of biosensors, super-resolution imaging, and novel light-emitting devices.