The fluorescence properties of AIE‐active N‐amidinated fluoroquinolones, efficiently obtained by a perfluoroaryl azide–aldehyde–amine reaction, have been studied. The fluorophores were discovered to elicit a highly sensitive fluorescence quenching response towards guest molecules with hydrogen‐bond‐donating ability. This effect was evaluated in a range of protic/aprotic solvents with different H‐bonding capabilities, and also in aqueous media. The influence of acid/base was furthermore addressed. The hydrogen‐bonding interactions were studied by IR, NMR, UV/Vis and time‐resolved fluorescence decay, revealing their roles in quenching of the fluorescence emission. Due to the pronounced quenching property of water, the N‐amidinated fluoroquinolones could be utilized as fluorescent probes for quantifying trace amount of water in organic solvents.