We have successfully created a graphene microroll aptasensor (GMA) through the self-folding of bilayer thin films. The inner surface of the GMA consists of a functionalized graphene surface, which works as a fluorescence aptamer-based biosensor. This is achieved by combining previously reported two-dimensional (2D) graphene aptasensor technology, a novel self-folding process for flexible, multilayered polymeric films. The aptasensing portion is composed of a graphene surface, which is functionalized with a pyrene–aptamer–dye probe. The three components of the probe work as linkers to the graphene surface, a protein recognition part, and a fluorescence detection tag, respectively. In this paper, we discuss the specialized fabrication process required to produce the GMA. By confirming the diffusion of target molecules through the hollow space of the GMA, we also show the ability of this device to detect concentration changes in real time.