A highly sensitive amperometric choline biosensor was fabricated using bamboo-like multi-walled carbon nanotubes (BCNTs)/ionic liquid (IL)/Prussian blue (PB) nanocomposite modified glassy carbon (GC) electrode. The PB nanoparticles were first electrodeposited on the surface of a BCNTs/IL/GC electrode. The Ni2+ ions were then electrochemically incorporated into the PB structure to improve its stability in mild alkaline media. Choline oxidase was next immobilized on the modified electrode using a cross-linking method. Amperometric measurements of choline were performed at 0.05V vs. Ag/AgClin 0.05 M phosphate buffer, pH 7.4. The choline biosensor exhibited a high sensitivity of 345.4 mu AmM-1 cm(-2) with a detection limit of 4.5 x 10(-7)M. The amperometric response was linear from 4.5 x 10(-7) to 1.0 x 10(-4)M. The proposed biosensor displayed a short response time of 2s, low K-m value of 9.7 x 10(-5) M, good storage stability and high selectivity. (C) 2014 Elsevier B.V. All rights reserved.