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Hydrogel biosensors usually suffer from a slow response, which severely hinders their practical applications. Here a new optical glucose biosensor was designed, using glucose-sensitive hydrogel films as both glucose-sensing material and Fabry-Perot cavity. The film was fabricated by layer-by-layer assembly from partially oxidized dextran (PO-Dex), chitosan, and glucose oxidase (GOD). The film responds to glucose because the incorporated GOD converts glucose to gluconic acid, and thus lowers the local pH in the film, and, in turn, triggers the pH-sensitive film to swell. The glucose-induced swelling causes a shift of Fabry?Perot fringes on the reflection spectra of the film, from which the glucose concentration can be reported. The new sensor works well under physiological conditions. Potential interferents, such as diols for phenylboronic acid-based sensors and electroactive compounds for electrochemical sensors, do not influence the new sensor. The sensor can respond reversibly over a wide range of glucose concentration. Particularly, it responds linearly within the clinically relevant glucose range (0–20 mM). More importantly, because the film is very thin, the new sensor can respond quickly, making it potential for real-time, continuous glucose monitoring.