DNA origami can be applied as a "ruler" for nanoscale calibration or super-resolution fluorescence microscopy with an ideal structure for defining fluorophore arrangement, allowing the distance between fluorophores to be precisely controlled at the nanometer scale. DNA origami can also be used as a nanotag with arbitrary programmable shapes for topological identification. In this study, we formed a hexagonal origami structure embedded with three different fluorescent dyes on the surface. The distance between each fluorescent block was ~120 nm, which is below the diffraction limit of light, allowing for its application as a nano-ruler for super-resolution fluorescence microscopy. The outside edge of the hexagonal structure was redesigned to form three different substructures as topological labels. Atomic and scanning force microscopy demonstrated the consistent nanoscale distance between morphological and fluorescent labels. Therefore, this fluorophore-embedded hexagonal origami platform can be used as a dual nanoruler for optical and topological calibration.