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In this work, a novel naked eye and fluorescence sensor for acetylcholinesterase (AChE) determination based on copper/silver nanoclusters (Cu/Ag NCs) was developed. L‐Histidine protected Cu/Ag NCs were prepared and exhibited superior properties compared with copper nanoclusters, with the quantum yield increased from 4.3% to 13.2%. The fluorescence of Cu/Ag NCs can be easily quenched by 2‐nitro‐5‐thiobenzoate anion (TNB) owing to the inner filter effect. TNB is the product between 5′‐Dithiobis‐(2‐nitrobenzoic acid) (DTNB) and thiols. However, AChE can specially catalyze acetylthiocholine (ATCh) to hydrolyze and release biothiols (TCh). Based on this, the determination of AChE was achieved by introducing DTNB. A naked eye sensor was also constructed owing to the color change. [ABSTRACT FROM AUTHOR]