We reported on the utilization of a novel attractive nanoscaled calcium carbonate (nano-CaCO(3))-polyphenol oxidase (PPO) biocomposite to create a highly responsive phenol biosensor. The phenol sensor could be easily achieved by casting the biocomposite on the surface of glassy carbon electrode (GCE) via the cross-linking step by glutaraldehyde. The special three-dimensional structure, porous morphology, hydrophilic and biocompatible properties of the nano-CaCO(3) matrix resulted in high enzyme loading, and the enzyme entrapped in this matrix retained its activity to a large extent. The proposed PPO/nano-CaCO(3) exhibited dramatically developed analytical performance such as such as a broad determination range (6 x 10(-9) -2 x 10(-5)M), a short response time (less than 12 s), high sensitivity (474 mA M(-1)), subnanomolar detection limit (0.44 nM at a signal to noise ratio of 3) and good long-term stability (70% remained after 56 days). In addition, effects of pH value, applied potential, temperature and electrode construction were investigated and discussed.