A base amount-dependent fluorescence enhancement-based strategy is put forward to determine vascular endothelial growth factor 165 (VEGF165) in human serum by the use of hairpin DNA-silver nanoclusters (hDNA-AgNCs) and oxidized carbon nanoparticles (CNPs). The hDNA-AgNCs aptasensing probe consists of AgNCs-contained hairpin loop (that generates a fluorescence signal), hairpin stem (that makes the structure stable), and the terminal aptamer 1 (that recognizes the target together with aptamer 2). It has been demonstrated that the fluorescence intensity of hDNA-AgNCs is ~ 3-fold stronger than that of single-stranded DNA-AgNCs (ssDNA-AgNCs), and hDNA-AgNCs have a strong dependence of fluorescence enhancement on the base amount in hairpin stem and loop. Upon the addition of oxidized CNPs, the terminal aptamer 1 of hDNA-AgNCs can adsorb onto the surface of oxidized CNPs via π-π stacking, and the fluorescence of hDNA-AgNCs (with excitation/emission maxima at 490/567 nm) is quenched via fluorescence resonance energy transfer (FRET). When aptamer 2 and VEGF165 are subsequently added, aptamer 1, VEGF165, and aptamer 2 reassemble into an intact tertiary structure, and the fluorescence is recovered because hDNA-AgNCs are far away from the surface of oxidized CNPs and the FRET efficiency decreases. Under the optimized conditions, the aptasensing probe can selectively assay VEGF165 with a detection limit of 14 pM. The results provide a label-free and sensitive method to monitor VEGF165 in human serum. Schematic representation of the strong dependence of fluorescence enhancement on base amount in stem and loop of hairpin DNA-silver nanoclusters. The probe can be used to assay vascular endothelial growth factor 165 (VEGF165) and give a judgment whether human serum VEGF165 is at a normal or abnormal level for clinical diagnosis.