The cytidine 5′-monophosphate (CMP)-protected gold nanoclusters (AuNCs@CMP) was developed to be a fluorescent nanosubstrate for direct ratiometric determination of alkaline phosphatase (ALP). The binding mode investigations revealed that the ligand CMP binds to the gold core via two oxygen atoms of ribose and cytosine and leaves the phosphate group freely outside, promoting AuNCs@CMP to be the direct nanosubstrate of ALP. AuNCs@CMP emits at 570?nm while the hydrolysis product of AuNCs@Cyt emits at 485?nm; the large emission gap between them provided a ratiometric approach for ALP determination. Moreover, a natural and biofunctional oligomer, chitosan oligosaccharide (COS), was introduced to the system, which significantly amplified the fluorescence signals of AuNCs@Cyt and improved the limit of detection (LOD) down to 0.00026?U·L^?1. Furthermore, the intrinsic mechanism of AuNCs@CMP hydrolysis by ALP and the COS amplification on AuNCs@Cyt were studied in-depth, which indicated the emission enhancement was attributed to the aggregation-induced emission enhancement (AIEE) property of the produced AuNCs@Cyt. Finally, the developed approach was successfully applied to determine ALP in human serum and the evaluation of ALP inhibitors. Therefore, the present study develops a novel nanosubstrate for ALP determination in a range of 0.0050–0.25?U·L^?1 and provides a straightforward way to amplify the fluorescence signal by employing available polymer. The work will stimulate and encourage the structural promotion of metal nanoclusters, and extend their biological applications broadly.