<p>A highly sensitive and stable fluorescent strategy for detecting alkaline phosphatase (ALP) in complex samples was developed using oxidized single-walled carbon nanohorns (oxSWCNHs) and exonuclease I (Exo I). A ssDNA probe, comprising a fluorophore-labeled aptamer and a 3′ phosphate group, was designed and synthesized. In the absence of ALP, the ssDNA probe binds to oxSWCNHs, causing fluorescence quenching. When ALP is present, it removes the 3′ phosphate group from the ssDNA, releasing a free 3′-OH group. The dephosphorylated ssDNA is then hydrolyzed by Exo I, generating a single base and FAM, which cannot bind to oxSWCNHs, resulting in enhanced fluorescence of the system. This strategy was successfully applied to image hepatocytes, showing the potential of our sensing system for ALP bioimaging in living cells. The method has good selectivity and high sensitivity under optimized experimental conditions, with a detection limit of 0.4&#xa0;mU/mL and a range of 0.5–50&#xa0;mU/mL. Additionally, it was used to study the inhibitory effects of Na₃VO₄. This method has great potential for the quantitative detection of ALP in clinical diagnostics.</p> Graphical Abstract <p></p>