Turn‐on fluorescence probes can visualize the enzyme activity with high contrast. We have established a new turn‐on mechanism, activator‐induced nucleophilic quencher detachment (AiQd), and developed AiQd‐based turn‐on fluorescence probes for the detection of enzymes. Herein, we demonstrate that the precise steric control efficiently quenches the fluorescence of AiQd‐based turn‐on probes before the enzymatic transformation. Theoretical calculation appropriately predicted the ratio of the fluorescence‐quenched closed‐ring form of probes. βC5S‐A, which has a sterically demanding methyl group at the β‐position of a fluorescence‐quenching nucleophilic mercapto group, showed a low background signal. βC5S‐A responded to aldehyde dehydrogenase 1A1 (ALDH1A1) with high selectivity, thereby enabling high‐contrast live imaging of cancer stem cells (signal‐to‐noise ratio >10). The ALDH1A1‐responsiveness of βC5S‐A was not significantly affected by amino acids and biological thiols, such as cysteine and glutathione.

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