A dual-stimuli responsive i-motif/nanoflare for molecule detection in lysosomes was designed. By combining the structure-switchable i-motif sequence and high recognition ability of an adenosine triphosphate (ATP) aptamer, subcellular sensing and visualization sensing of ATP in lysosomes at the subcellular level can be achieved. This general sensing technique can be applied for a broad range of cellular communication studies to improve our understanding of subcellular signaling and function.