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Glutathione (GSH) is the most abundant biothiol in cells and plays key roles in biological processes. Therefore, it is vitally important to detect GSH in biological samples. In this work, by using a novel quantum dots-based “off-on” fluorescent lateral flow test strip (LFTS), the authors describe a rapid and sensitive biosensor for point-of-care testing of GSH. In this design, bovine serum albumin (BSA) functionalized quantum dots (QDs) are chosen as signal reporter and modified on the test line of the LFTS. When Ag + binds to the surface of QDs, the fluorescence of QDs can be quenched efficiently owing to the electron transfer from QDs to Ag + . In the presence of GSH, due to the much stronger affinity between Ag + and GSH, Ag + prefers to react with GSH to form Ag + -S bond. Consequently, the fluorescence of QDs is restored and GSH quantification is realized successfully. Under the optimal conditions, a good linear relationship from 0.1 μM to 15 μM with a detection limit of 25 nM was obtained, and the whole process can be accomplished quickly within 10 min. In addition, this proposed biosensor showed acceptable recovery (98.4%–102.2%) for the detection of GSH in Hela cells extract samples. [ABSTRACT FROM AUTHOR]