A fluorescent sensor for alkaline phosphatase (ALP) was realized by covalently immobilizing fluorescein molecules onto the surface of silicon nanowires (SiNWs) and phosphorylation of the immobilized fluorescein group. Based on the fluorescence enhancement, the new type of sensor exhibited good selectivity and sensitivity to ALP, and displayed a linear relationship between the fluorescence intensity and the ALP concentration in the range of 0.0175–0.3 U/mL. A single nanowire-based sensor was successfully employed to monitor the ALP activity and examine the inhibition effect of levamisole on the ALP activity. The fluorescence images of the single nanowire-based sensor showed a high spatial resolution. The present sensor paves a way to directly assay the activity of intracellular ALP by inserting a single nanowire-based sensor into a cell.