Abstract Herein, a dual‐signal‐output glass nanopore system was developed for sensing glucose. Upon binding glucose, the boronic acid‐containing stimuli‐responsive polymer underwent a wettability switch and pKa shift. When the polymer was immobilized on the inner wall of nanopore, the nanopore offered a sensitive method for evaluating glucose by ion rectification. Besides, due to the electrostatic assembly of cationic pyrene onto the glucose‐bound anionic polymer, the pyrene excimer emission was observed. By separating the substrate binding and fluorescence reporting process, this work avoided the fluorescence labeling of the target and nanopore, thus simplifying the design of dual‐signal‐output nanopore for potential point‐of‐care test.
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