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Bitterness elicits unpleasant sensations in humans, which can hinder the acceptance of foods and medication adherence. Therefore, identifying and masking bitter tastes is crucial for developing palatable foods and promoting medication compliance in the food and pharmaceutical industries. To achieve this, employing agonism and antagonism of bitter taste receptors as effective strategies at the molecular level is essential. In this study, a bioelectronic tongue is developed to characterize the agonism and antagonism of bitter taste receptors. The human bitter taste receptors hTAS2R16 and hTAS2R31 are produced using an Escherichia coli expression system and reconstituted into nanodiscs (NDs). Subsequently, hTAS2R16‐ and hTAS2R31‐NDs are immobilized on the surface of graphene field‐effect transistors (FETs) to construct bioelectronic tongues. The developed system sensitively detected the bitter agonists, salicin and saccharin, at concentrations as low as 100 fM, with high selectivity in real‐time. The dose‐dependent curves shifted and K values decreased by the antagonists of hTAS2R16 and hTAS2R31, indicating antagonism‐based masking of bitter taste. Therefore, the developed bioelectronic tongue holds promise for identifying bitter tastes and evaluating the masking of bitterness based on the agonism and antagonism of hTAS2Rs. [ABSTRACT FROM AUTHOR]