Optical sensors based on discrete plasmonic nanostructures are very attractive for probing biomolecular interactions at the single-molecule level and have been applied as single nanoparticles or plasmonic rulers or reconfigurable multi-nanoparticle assemblies. However, their adaptation as a versatile sensing platform is limited by the research-grade instrumentation required for single-nanostructure imaging and/or spectroscopy and complex data fitting and analysis. Additionally, the dynamic range is often too narrow for the quantitative analysis of targets of interest in biodiagnostics, food safety or environmental monitoring. Herein we present plasmonic assembly comprising a core nanoparticle surrounded by multiple layers of satellite nanoparticles through aptamer linker. The layer-by-layer assembly of the satellite nanoparticles yields uniform discrete nanoparticle clusters on a substrate with enhanced optical properties. Binding of the model target (adenosine 5’-triphosphate, ATP) induces disassembly and leads to a dramatic decrease in the scattering intensity that can be analyzed readily from darkfield images. The sensing performance of assemblies, such as detection limit, dynamic range and sensitivity, can be tuned by controlling the size of the assembly. Surprisingly, the substrate-anchored nanoparticle assemblies are selective to only ATP, and not other adenine-containing compounds such as AMP and ADP at concentration less than 50 mM. It presents an approach to increase the specificity of the aptamer, which otherwise binds to all adenine-containing compounds if in free-form in solution. By assembling the clusters on a flexible support, cellular ATP can be directly detected by lysing adherent cells in close contact with the sensor – a process that does not require any sample preparation or purification. Enhancing the optical detection signal via designing and engineering nanoparticle assemblies could enable their use with low-cost portable imaging systems and broaden their applicability beyond the study of biomolecular interaction.