This study demonstrates a novel carbon nanotubes-hydroxyapatite (CNTs-HA) nanocomposite-based compartment-less glucose/O2 biofuel cell (BFC) with the glucose oxidase (GOD) as the anodic biocatalysts and the laccase as the cathodic biocatalysts. CNTs-HA nanocomposite prepared by the self-assembly method via an aqueous solution reaction has been used as the co-immobilization matrix to incorporate biocatalysts, i.e. GOD and laccase successfully. Moreover, the three-dimensional configuration of the CNTs-HA films electrode would be advantageous to the glucose oxidation on the bioanode and O2 electroreduction on the biocathode of BFC. The maximum power density delivered by the assembled glucose/O2 BFC could reach 15.8 Wcm-2 at a cell voltage of 0.28V with 10mM glucose. The results indicate that the CNTs-HA nanocomposite is believed to be very useful for the development of novel BFCdevice.