The dysfunction of acetylcholinesterase (AChE) activity is responsible for many complex nervous diseases, demonstrating the urgent demand of constructing assays with robust performance for determining AChE activity. Herein, a ratiometric fluorescent (RF) sensing platform for AChE activity was strategically fabricated, which was based on the self-assembly of Ag+-modified Au nanoclusters and silicon nanoparticles (Ag-AuNCs@SiNPs) nanohybrids to perform as the RF probes. The Ag-AuNCs@SiNPs nanohybrids were facilely prepared through the electrostatic attraction between positively charged SiNPs and negatively charged Ag-AuNCs. AChE can catalyze the hydrolysis of ATCh into thiocholine (TCh) that in turn quenched the fluorescence of Ag-AuNCs in the Ag-AuNCs@SiNPs nanohybrids. Meanwhile, the fluorescence emission intensity of SiNPs remained stable. Consequently, based on the change of RF signal, AChE activity was sensitively detected in the range of 0.03–4.0 U/L with a low detection limit of 0.0021 U/L. This RF strategy was applied for AChE activity sensing in human serum sample with reliable performance, indicating the vast application prospect of this assay for the AChE-related clinical investigation.