An electrochemical biosensor based on a carbon nanotube (CNT) layer with glucose oxidase (GOx) enzyme sandwiched between 6-nm-thick acetonitrile plasma-polymerized films (PPFs) was fabricated. The lower PPF was deposited onto a sputtered gold electrode. The optimization of the casting formation of the CNT layer onto the lower PPF is addressed. As a result, the best candidate for the casting solution was a 1:1 mixture of phosphate buffer and ethanol, and the optimized CNT concentration was 7.5 mg ml–1. The optimized glucose biosensor characteristics exhibited ultrasensitivity (a sensitivity of 11 μA mM–1 cm–2 (4.9–19 mM), a detection limit of 6.2 μM at S/N = 3, +0.8 V vs Ag/AgCl), and a rapid response (< 4 s to reach 95% of maximum response). This high performance is attributed to the excellent electrocatalytic activity and enhancement of electron transfer that CNTs provide, and because the PPF and/or plasma process for CNTs are an enzyme-friendly platform.