This work describes for the first time signal‐on electrochemiluminescence (ECL) enzyme biosensors based on cadmium sulfide nanocrystals (CdS NCs) formed in situ on the surface of multi‐walled carbon nanotubes (MWCNTs). The MWCNT–CdS can react with H2O2 to generate strong and stable ECL emission in neutral solution. Compared with pure CdS NCs, the MWCNT–CdS can enhance the ECL intensity by 5.3‐fold and move the onset ECL potential more positively for about 400 mV, which reduces H2O2 decomposition at the electrode surface and increases detection sensitivity of H2O2. Furthermore, the ECL intensity is less influenced by the presence of oxygen in solution. Benefiting from these properties, signal‐on enzyme‐based biosensors are fabricated by cross‐linking choline oxidase and/or acetylcholine esterase with glutaraldehyde on MWCNT–CdS modified electrodes for detection of choline and acetylcholine. The resulting ECL biosensors show wide linear ranges from 1.7 to 332 µM and 3.3 to 216 µM with lower detection limit of 0.8 and 1.7 µM for choline and acetylcholine, respectively. The common interferents such as ascorbic acid and uric acid in electrochemical enzyme‐based biosensors do not interfere with the ECL detection of choline and acetylcholine. Furthermore, both ECL biosensors possess satisfying reproducibility and acceptable stability.

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