Polyaniline-encapsulated carbon/copper composite nanofibers (PANI/CuCNFs) were synthesized by a facile and efficient method combining electrospinning and in-situ polymerization, which were further applied to the laccase (Lac) based biosensor for polyphenol pollutant detection. The morphology and composition of the fabricated nanocomposite were investigated by a series of advanced characterization techniques. The electrochemical behaviour of the novel biosensor was studied through cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry. Results showed that the addition of PANI/CuCNFs apparently improved the electrocatalytic properties of the Lac-based biosensor towards hydroquinone. The constructed biosensor possessed a high sensitivity (41.65 mu A mM(-1)), a low detection limit (0.24 mu M), and a wide linear range from 500 nM to 110 mu M, as well as satisfied selectivity, reproducibility and repeatability. Moreover, the biosensor gave a successful application in hydroquinone measurement of river water. This work suggested that the Lac biosensor modified by PANI/CuCNFs has a great potential in the application of phenolic pollutants detection.