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• A novel polyphenol biosensor based on hybrid assemblies of polyaniline-polyacrylonitrile-graphene has been fabricated. • The excellent smooth steady-state currents were obtained to avoid errors caused by the current drifting and fluctuation. • The proposed biosensor exhibited high sensitivity and selectivity for determination of phenolic compounds in water samples. Phenolic compounds are major environmental pollutants due to their toxic and hazardous nature on human health. A fast, sensitive and stable sensor for determination of phenolic compounds in the environmental water remains challenging. Herein, a biosensor platform with stable response current was fabricated by entrapment of polyphenol oxidase (PPO) into hybrid assemblies of the conducting polyaniline (PAni)-porous polyacrylonitrile (Pan)-nanostructured graphene (GRA) and phase inversion process. The porous structure of Pan provided a favorable microenvironment for easily binding to PAni and GRA to obtain hybrid assemblies for effective immobilization of enzyme and increased synergistic effect. The morphologies and the electrochemical behaviors of the as-prepared biosensor were investigated using scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively. The proposed biosensor showed excellent sensitivity (6.46 μA μM−1 cm−2) and fast response time (˜5 s) with low detection limit (2.65×10−7 M) under the optimal pH value and applied potential. The biosensor was highly selective towards p-cresol that almost no signal was detected from common interferents. The biosensor was used for determination of phenolic compounds in water samples with satisfactory results compared with that of UPLC, demonstrating its great potential as a biosensor for the rapid determination of phenolic pollutants. [ABSTRACT FROM AUTHOR]