A novel amperometric biosensor utilizing carbon nanotube (CNT)–Ni nanoparticle hybrid arrays for ethanol detection has been developed. The sensor was fabricated by e-beam deposition of thin Ni film on arrayed multi-walled carbon nanotubes grown directly on Si substrate. As the Ni film thickness is ≤200 nm, high-resolution SEM and TEM images showed well-dispersed and strongly adhered crystalline Ni nanoparticles uniformly populated on the sidewalls of CNTs, and the sensitivity of the resultant CNT–Ni sensor generally increased with increasing nickel thickness. The best sensor showed a wide response range of 50–600 μM, a response time of <10 s, and a sensitivity of 14.81 μA μM −1 cm −2 which is ∼380% higher in comparison with the nanostructured Ni/Pt/Ti electrodes.