Abstract An electrochemiluminescent (ECL) choline biosensor was developed by the drop coating of choline oxidase (ChOx) onto a carbon nanotubes (CNTs)/potassium ferricyanide (K 3Fe(CN) 6)-modified platinum electrode with ECL of luminol as readout signal. CNTs improved the loading capacity of enzyme, as well as the biocompatibility and electron transfer of the electrode surface, whereas K 3Fe(CN) 6 facilitated activation of the enzyme and enhancement of the ECL signal, so the developed biosensor exhibited excellent analytical performance. The biosensor gave optimal results while the Pt electrode was modified with 4 μL of 0.33 g L −1 CNTs dispersoid, 2 μL of 0.1 M K 3Fe(CN) 6 and 1.5U of ChOx. In the PBS buffer (pH 7.4) containing 8 × 10 −6 M luminol, the ECL signal linearly responded to the concentration of choline from 1 × 10 −7 M to 4 × 10 −3 M ( r = 0.994) with a detection limit of 1.21 × 10 −8 M under 30 (C. The developed biosensor was applied to measure the concentration of choline in rat blood sample and the result was satisfied, with an average recovery of 101.1%. The as-prepared biosensor showed good stability and repeatability.