In this study, a facile and fast aqueous-phase synthetic method for preparing chitosan-based fluorescent nanogels (CS) by exploiting the aggregation-induced emission (AIE) property of copper nanoclusters (Cu NCs) is proposed. Owing to the spatial confinement provided by the crosslinking network structure in chitosan-based nanogels and the electrostatic interaction between positively charged chitosan nanogels and negatively charged Cu NCs, the fluorescent intensity of the as-prepared CS-Cu NCs was increased by approximately 17-fold compared with that of Cu NCs. The fluorescence quantum yield is increased by more than four times; the as-prepared CS-Cu NCs exhibited a quantum yield of 64.12%. Additionally, CS-Cu NCs exhibited significantly improved stability in aqueous solution, including excellent oxidation resistance, high anti-salt stability, good thermal stability, and enhanced capacity of anti-photobleaching. These properties provide a fundamental guarantee for the application of Cu NCs with AIE property in the biosensor and bioimaging. Upon the addition of the anticancer drug methotrexate (MTX) to CS-Cu NCs, their fluorescent intensity was markedly quenched. Based on the observed fluorescence-quenching phenomenon of CS-Cu NCs induced by MTX, a novel fluorescence quenching nanoprobe was designed for detecting the labeling amount percentage of commercially available methotrexate tablets. The experimental results validated that our proposed nanoprobe exhibits a wider dynamic linear range and excellent accuracy with a low limit of detection (LOD) of 4.16 μM, thereby expanding the potential application of chitosan-based nanogels encapsulating metal nanoclusters presenting AIE property in pharmaceutical quality control.