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A novel biosensor based on gold nanoparticles dispersed in ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (Au-BMI.PF 6 ) and polyphenol oxidase (PPO) from garlic (Allium sativum L.) immobilized in clay was developed for determination of polyphenol by square-wave voltammetry. Under optimized conditions, the analytical curve showed a linear range for esculetin concentrations from 0.40 to 9.86 mM (R = 0.9997), with a limit of detection (LOD) of 0.11 mM. This biosensor demonstrated good repeatability and reproducibility, with relative standard deviations of 3.5 % and 4.6 %, respectively. The method was successfully applied to the determination of esculetin content in black tea samples.