We report the successful construction of plasmonic core-satellite nanostructured assemblies on two-dimensional substrates, based on a strategy of combining DNA-functionalized plasmonic nanoparticles (NPs) with the specific recognition ability toward target to enable satellite NPs to self-assemble around the core immobilized on substrates. A strongly coupled plasmonic resonance band was observed because of the close proximity between core and satellite NPs, which presented significant red-shift and enhanced extinction with respect to the local surface plasmon resonance (LSPR) band of individual core NPs on the substrate. The functionality of this core-satellite nanostructured assembly as a biosensor was further explored, and the changes in extinction intensity and the peak shift of the plasmonic coupling resonance band arising from the probe-target DNA binding event all proved to be useful criteria for target DNA detection. Moreover, high selectivity down to single-base mismatched DNA was achieved using this strongly coupled plasmonic core-satellite nanostructured assembly on a substrate. Such substrate-based detection was advantageous, and its reusability and high cycle stability were demonstrated after five cycles of disassembly and reassembly. Our work demonstrates the biosensing capacity of this DNA-functionalized plasmonic nanoassembly model system on two-dimensional substrate, which is also applicable to the detection of numerous DNA-recognized biomolecules. Likewise, the presented construction method can be extended to fabricate other compositional core-satellite nanoassemblies.