To increase the protein capability and simplify biosensing fabrication, a three-dimensional (3D) nitrogen-doped carbon nanotubes (NCNTs) on free-standing electrospun carbon nanofibers film (NCNT@CNFs) was prepared via pyrolysis of pyridine. The density, size and nitrogen content of the "quasi-aligned" NCNT arrays can be easily controlled by modulating the growth temperature. By directly dropping the free-standing and flexible composite onto the electrode surface with high glucose oxidase loading (3.2 x 10(-9) mol cm(-2)), a glucose biosensor with remarkable sensitivity (24.8 mAM(-1) cm(-2)), a low detection limit (6 mu M at a signal-to-noise ratio of 3), and a wide linear range can be simply constructed. In addition, the biosensor exhibited high reproducibility, good storage stability, satisfactory anti-interference ability, and strong applicability to real samples. The N-doping combining with abundant defective sites and the favorable 3D network structure could effectively avoid enzyme leakage, maintain the bioactivity of the enzyme, and facilitate electron transfer, which resulted in excellent electrocatalytic performance. The facile preparation method and attractive analytical performance make this robust electrode material promising for the development of effective electrochemical sensors. (C) 2015 Elsevier B.V. All rights reserved.