<p>Although fluorescent probes have been used extensively for alkaline phosphatase (ALP) sensing, most involve a tedious, prolonged, or high-cost synthesis procedure. Thus, it is still necessary to develop convenient and economical fluorescent probes for sensitive determination of ALP. Herein, a facile fluorescent strategy was designed for ALP detection utilizing the fluorogenic response of thiamine (TH) triggered by Cu²⁺ (Cu²⁺-TH system). It was found that Cu²⁺ facilitated the oxidation of non-fluorescent TH to fluorescent thiochrome (TC) in an alkaline environment, while pyrophosphoric acid (PPi) interacted with Cu²⁺ to form PPi-Cu²⁺ chelates, preventing the oxidation of TH to fluorescent TC. Consequently, a faint fluorescence output was detected in the system. After adding ALP into this system, PPi was catalyzed to phosphate (Pi), which combined weakly with Cu²⁺. A large amount of TH was oxidized into fluorescent TC by free Cu²⁺, leading to high fluorescence. Therefore, an ALP sensing platform was constructed by utilizing the fluorogenic reaction of the Cu²⁺-TH system as the signal reporter. The assay exhibited favorable analytical performance for ALP measurement. A linear correlation was obtained between the ALP concentration (0.01–1&#xa0;mU/mL) and the fluorescence intensity of the Cu²⁺-TH system, with a limit of detection as low as 0.0038&#xa0;mU/mL. Additionally, the assay was further employed for analyzing ALP content in complex biological samples. This work thus advances the science of in situ fluorogenic reaction in the field of biosensing.</p> Graphical Abstract <p></p>