Fluorescence-based probes and sensors have emerged as indispensable tools for the detection of alkaline phosphatase (ALP), a clinically significant enzyme, owing to their high sensitivity, real-time imaging capabilities, and superior spatial resolution. This review examines fluorescence-based ALP detection techniques published between 2020 and 2025 and divides them into three main categories, which are small-molecule fluorogenic probes, nanomaterial-based fluorescent sensors and nucleic acid-assisted hybrid systems. In this context, particular attention is given to the structural evolution of various fluorophore scaffolds and the engineering of molecular dye platforms designed to modulate specific photophysics for enzyme-responsive signaling. By utilizing cutting-edge design components like near-infrared and ratiometric fluorophores, nanoparticle-based signal amplifiers, and deoxyribonucleic acid/ribonucleic acid (DNA/RNA)-mediated amplification circuits (including aptamer and clustered regularly interspaced short palindromic repeats (CRISPR)-driven mechanisms), researchers have improved analytical performance across these platforms. A wide range of applications, such as clinical diagnostics (ultrasensitive serum assays), live-cell and in vivo imaging of enzyme activity, environmental monitoring, and high-throughput screening. These advances have enabled applications ranging from ultrasensitive serum assays to live-cell imaging and portable readouts. This review highlights the increasing significance of fluorescence-based ALP detection in analytical chemistry and biomedicine by showcasing both state-of-the-art probe design and practical deployment. It also explores how current developments are laying the groundwork for next-generation ALP sensors with even higher sensitivity, specificity, and versatility. In addition to summarizing recent reports, we benchmark the 2020–2025 literature against pre-2020 ALP sensing paradigms to delineate genuine step-changes and to highlight persistent limitations that remain unresolved. Lastly, it discusses the opportunities and challenges that still need to be overcome before these ALP sensing technologies can be used in clinical and field settings.
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