<p>Ultra-stable copper nanoclusters were fabricated for the sensitive determination of cholesterol based on fluorescence turn-off. Red-emitting copper nanoclusters capped with cysteine were first synthesized with a short storage time (15 d). Then, the addition of chitosan and the adjustment of media pH were applied to modulate the unstable clusters into exceptionally stable aggregates with cyan-green emission possessing a long lifetime over 180&#xa0;days. The aggregation-induced emission (AIE) of chitosan-modified copper nanoclusters (Cys-CuNCs) can be significantly quenched by cholesterol due to the interaction between cholesterol and chitosan. The fluorescence response of Cys-CuNCs to cholesterol exhibits a rapid equilibrium (1&#xa0;min), high sensitivity with a detection limit of 19.63&#xa0;μmol·L⁻¹ (3σ, <i>n</i> = 3), and a wide linear concentration range of 25.0 ~ 1000.0&#xa0;μmol·L⁻¹. The proposed method has good stability and can be employed for cholesterol determination in biofluids.</p> Graphical Abstract <p></p>