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A general strategy for design and fabrication of nanoscale fluorescent sensors using electrostatic layer-by-layer assembly is presented. Using the approach of depositing oppositely-charged species onto colloidal micro/nanotemplates, two sensor systems employing fluorescent indicators and functional biomolecules are proposed: thin sensor films on solid carriers, and micro/nanocapsules containing sensing chemistry. Results demonstrating these concepts are described for ion (sodium and potassium), oxygen, and glucose sensors. The work suggests great potential for layer-by-layer architecture to fabricate nanostructured fluorescent sensors using nanoscale solid carriers and hollow microshells.