ZnO loaded carbon nanofibers (ZnO/CNFs) were successfully fabricated by a combination of electrospinning, carbonization and a hydrothermal process. A novel biosensor was fabricated based on a composite of ZnO/CNFs, laccase (Lac), and Nafion. The addition of ZnO/CNFs apparently facilitated the direct electron transfer (DET) between the active center of Lac and the surface of the glassy carbon electrode (GCE). A pair of stable and well-defined redox peaks was observed on the Nafion-Lac-ZnO/CNF modified GCE. Meanwhile, square wave voltammetry (SWV) was employed to investigate the biosensor, and the sensor showed highly efficient electrocatalysis toward hydroquinone with a sensitivity of 28.50 mu A mu M-1, a detection limit of 9.50 nM (S/N = 3), a linear range from 5.00 x 10(-7) to 2.06 x 10(-6) M, as well as good selectivity and stability. Furthermore, this novel biosensor was successfully used in detecting hydroquinone in real water samples.