Alkaline phosphatase (ALP) is ubiquitous in mammalian tissues, facilitating the dephosphorylation of diverse biomolecules. As a key biomarker, ALP activity monitoring is of great significance for clinical research and disease diagnosis. Here, a sensitive and portable fluorescent fiber optic sensor based on silicon quantum dots (SiQDs) was developed for detecting ALP activity. Green-fluorescent SiQDs were synthesized using a one-pot hydrothermal method. A tapered optical fiber was facilely modified with SiQDs through in situ hydrogel polymerization reaction. The hydrogel-based optical fiber ALP sensor was meticulously designed by incorporating ALP-triggered cascade reaction. ALP catalyzes the substrate hydrolysis to produce ascorbic acid (AA), which then triggers a chromogenic redox reaction between AA and Cu(II)-neocuproine chelate to form highly colored Cu(I)-chelate, effectively quenching the fluorescence of the SiQDs due to inner filter effect. This method provides a linear detection of ALP activity within the range of 0.02–2.0 U/L. The sensor was applied to ALP activity detection in real serum samples, demonstrating its practicability and reliability. This sensor offers the advantages of high sensitivity (a detection limit of 0.015 U/L), exceptional selectivity, good practicability and simplified fabrication, presenting a portable and efficient technique for on-line ultrasensitive detection of ALP activity in biological samples. It provides a new perspective for developing high-performance optical fiber sensors, with significant practical value for applications in health monitoring, clinical diagnostics, and emergency analysis.