Enzymes-based glucose sensors are used as blood glucose sensors in diabetes treatment because of their high selectivity. The enzyme electrode of this sensor was obtained by immobilizing an enzyme on the electrode. However, it is difficult to stabilize an enzyme as a biological material. In this study, glucose oxidase (GOD), an enzyme that degrades glucose, was immobilized on the surface of cup-stacked carbon nanofilaments (CNF) with oxygen-containing functional groups on the surface. The formation of an amide bond between the amino group of GOD and the carboxyl group of the CNFs was expected to strongly immobilize GOD on the CNF surface. The use of CNFs with a large number of carboxyl groups as a support material for enzyme immobilization leads to improved glucose decomposition activity.